Camelid single-domain antibody directed against phosphorylated tau proteins and methods for producing conjugates thereof

ABSTRACT

The present invention relates to variable domains of a camelid heavy-chain antibodies directed against a phosphorylated tau protein and conjugates thereof. The present invention also relates to the use of these domains or conjugates for treating or diagnosing disorders mediated by neurofibrillary tangles, neuropil threads or dystrophic neurites, such as tauopathies.

The present invention relates to antibodies directed to phosphorylatedtau proteins and conjugates thereof. The present invention also relatesto the use of these antibody conjugates for treating or diagnosingdisorders mediated by phosphorylated tau proteins.

About 70% of all cases of dementia are due to Alzheimer's disease (AD)which is associated with selective damage of brain regions and neuralcircuits critical for cognition. Alzheimer's disease is characterized byneurofibrillary tangles (NFTs) in particular in pyramidal neurons of thehippocampus and numerous amyloid plaques.

Ultrastructural studies on AD brain specimens have revealed that NFTsare primarily composed of paired helical filaments (PHFs), and thesefilaments are pairs of axially opposed fibrils of approximately 10 nm indiameter with a helical tridimensional conformation at a regularperiodicity of 65 nm (Kidd 1963 Nature, 197, 192-3; Wiśniewski et al.1976 J Neurol Sci., 27, 173-81). In 1985, Brion et al. (Archives debiologie (Bruxelles), 95, 229-235) demonstrated that the major componentof the PHFs is the protein tau, a microtubulin associated protein (MAP).This result was confirmed by several authors: Grundke-Iqbal et al. 1986Proc Natl Acad Sci U.S.A., 83, 4913-7; Kosik et al. 1986 Proc Natl AcadSci U.S.A., 83, 4044-8; Delacourte and Defossez 1986 J Neurol Sci., 76,173-86; Wood et al. 1986 Proc Natl Acad Sci U.S.A., 83, 4040-3; Nukinaand Ihara 1986 J Biochem., 99, 1541-4; Pollock et al. 1986 Lancet, 2,1211; Montejo de Garcini et al. 1986 Biochem Biophys Res Commun., 141,790-6. It was then shown that the protein tau in NFTs was abnormallyphosphorylated (Grundke-Iqbal and Iqbal, et al. 1986 Proc Natl Acad SciU.S.A., 83, 4913-7; Ihara et al. 1986 J Biochem., 99, 1807-10; Iqbal etal. 1986 Lancet, 2, 421-6; Brion et al. 1986 Lancet, 2, 1098; Köpke etal. 1993 J Biol Chem., 268, 24374-84), that leads to sequestration ofnormal tau and other MAPs (Alonso et al. 1994 Proc Natl Acad Sci U.S.A.,91, 562-6; Alonso et al. 1997 Proc Natl Acad Sci U.S.A., 94, 298-303),which causes disassembly of microtubules and thus impaired axonaltransport, normal neuronal and synaptic functions, leading to loss ofsynapses and death of neurons. Hyperphosphorylated tau also becomesinsoluble and self-aggregates into paired helical filaments (PHFs) andNFTs. The end of all these processes is dementia. Tau phosphorylation isregulated by the balance between multiple kinases and phosphatases(Iqbal et al. 2005 Biochim Biophys Acta, 1739, 198-210; Blennow et al.2006 Lancet, 368, 387-403). With the increased amount of the abnormallyphosphorylated tau, tau levels in the AD brain are around eightfoldhigher than in age-matched controls (Khatoon et al. 1992 J Neurochem.,59, 750-3). Whether tau and NFT formation are a cause or consequence ofAD is not currently known (Blennow et al. 2006 Lancet, 368, 387-403).

Tau was first isolated and identified as a MAP in the 70's (Weingartenet al. 1975 Proc Natl Acad Sci U.S.A., 72, 1858-62; Cleveland et al.1977 J Mol Biol., 116, 227-47; Cleveland et al. 1977 J Mol Biol., 116,207-25) and is mainly expressed in neurons (Tucker 1990 Brain Res BrainRes Rev., 15, 101-20; Schoenfeld and Obar 1994 Int Rev Cytol., 151,67-137). The human tau gene is located on chromosome 17 where itoccupies over 100 kb and contains at least 16 exons (Neve et al. 1986Brain Res., 387, 271-80; Andreadis et al. 1992 Biochemistry, 31,10626-33; Andreadis et al. 1995 Nucleic Acids Res., 23, 3585-93). Byalternative splicing, the tau gene yields different mRNA and results inthe production of six tau isoforms (Goedert et al. 1989 Neuron, 3,519-26; Himmler et al. 1989 Mol Cel Biol., 9, 1381-8). The six tauisoforms differ from one another by the presence of three or fourmicrotubule binding repeats (R) of 31-32 amino acids each and of one,two, or zero amino terminal inserts (N) of 29 amino acids each, givingrise to 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R taus. The six isoforms oftau protein range from 352 to 441 amino acids (Goedert et al. 1989Neuron, 3, 519-26; Himmler et al. 1989 Mol Cell Biol., 9, 1381-8;Goedert et al. 1989 The EMBO J., 8, 393-9). Each of these isoforms mayexert specific physiological functions since they are differentiallyexpressed during development. For example, 0N3R tau is the only isoformpresent in fetal brain while all six isoforms are expressed duringadulthood (Kosik et al. 1989 Neuron, 2, 1389-97; Goedert and Jakes 1990EMBO J., 9, 4225-30; Buée et al. 2000 Brain Res Brain Res Rev., 33,95-130).

The 4R:3R tau ratios for both mRNA and protein are approximately equalin normal brain, but disturbances usually increase these ratios in mostof the neurodegenerative tauopathies (Hanger et al. 2009 Trends MolMed., 15, 112-9). Using specific mAb, thehyperphosphorylated/pathological tau proteins can be visualized onWestern blots as bands between 55 and 74 kDa (Buée et al. 2000 Brain ResBrain Res Rev., 33, 95-130).

The tau441 (441 amino-acids) contains 80 serine/threonine and fivetyrosine putative phosphorylation sites and an established function oftau is binding to microtubules through its microtubule-binding domains(R), thereby promoting microtubule assembly and stability (Weingarten etal. 1975 Proc Natl Acad Sci U.S.A., 72, 1858-62; Cleveland et al. 1977 JMol Biol., 116, 227-47; Cleveland et al. 1977 J Mol Biol., 116, 207-25;Bihm et al. 1990 Acta Histochem Suppl., 39, 357-64; Nixon and Sihag 1991Trends Neurosci., 14, 501-6; Drechsel et al. 1992 Mol Biol Cell., 3,1141-54; Brandt and Lee 1993 J Neurochem., 61, 997-1005). Themicrotubule assembly-promoting activity of tau is regulated by itsdegree of phosphorylation. Hyperphosphorylation suppresses the abilityof tau to stimulate microtubule assembly (Lindwall and Cole 1984 J BiolChem., 259, 5301-5). AD hyperphosphorylation of tau at over 40serine/threonine sites has been identified (Hasegawa et al. 1992 J BiolChem, 267, 17047-54; Morishima-Kawashima et al. 1995 J Biol Chem., 270,823-9; Hanger et al. 1998 Trends Mol Med, 15, 112-9; Vega et al. 2005Brain Res Brain Mol Res., 138, 135-44). Phosphorylation of Ser202, theepitope recognized by AT8 mAb, was found to correlate with tangleformation and is reported to be an early marker of tau pathology ofAD-type (Mercken et al. 1992 Acta Neuropathol., 84, 265-72; Biernat etal. 1992 EMBO J., 11, 1593-7; Su et al. 1994 Neuroreport, 5, 2358-62;Hernandez et al. 2003 Neurob Aging., 24, 1087-94; Blazquez-Llorca et al.2011 J Alzheimer's Dis., 26, 683-98). Phosphorylation of Ser422 near theC-terminus of tau is also reported to associate closely with developmentof NFTs. The amount of tau phosphorylated at Ser422 was found toincrease with severity of AD and this phosphorylation site is also aprominent pathology in AD (Bussière et al. 1999 Acta Neuropathol., 97,221-30; Augustinack et al. 2002 Acta Neuropathol., 103, 26-35; Haase etal. 2004 J Neurochem., 88, 1509-20; Pennanen and Gotz 2005 BiochemBiophys Res Commun., 337, 1097-101; Deters et al. 2008 Eur J Neurosci.,28, 137-47; Grueninger et al. 2011 Mol Cell Biochem., 357, 199-207).

Unlike amyloid-beta (Aβ) deposits, NFTs develop in a stereotypical andpredictable pattern in AD brain (Arnold et al. 1991 Cereb Cortex, 1,103-16; Braak and Braak 1991 Acta Neuropathol., 82, 239-59; Braak et al.2006 Acta Neuropathol., 112, 389-404). Braak and Braak (1991 ActaNeuropathol., 82, 239-59) proposed six stages to describe progression ofNFTs in their clinicopathological study. The first NFTs consistentlyappear in the transentorhinal (perirhinal) region (stage I) along withthe entorhinal cortex proper, then spread in the CA1 region of thehippocampus (stage II). Next, NFTs develop and accumulate in limbicstructures such as the subiculum of the hippocampal formation (stageIII) and the amygdala, thalamus, and claustrum (stage IV). Finally, NFTsappear in all isocortical areas (isocortical stage), with theassociative areas being affected prior and more severely (stage V),followed by the primary sensory, motor, and visual areas (stage VI). Asevere involvement of striatum and substantia nigra can occur during thelate isocortical stage (Serrano-Pozo, Frosch, et al. 2011 Cold SpringHarb Perspect Med., 1, a006189). Multiple clinicopathological studiesfrom different groups have established that NFT burden in the braincorrelates with the severity and the duration of dementia (Arriagada etal. 1992 Neurology, 42, 631-9; Bierer et al. 1995 Arch Neurol., 52,81-8; Gómez-Isla et al. 1997 Ann Neurol., 41, 17-2; Giannakopoulos etal. 2003 Neurology, 60, 1495-500; Ingelsson et al. 2004 Neurology, 62,925-31). The selective distribution of NFTs described above matches withthe hierarchical neuropsychological profile typical of the AD-typedementia syndrome (Serrano-Pozo et al. 2011 Cold Spring Harb PerspectMed., 1, a006189).

In Alzheimer's disease (AD), accumulation of hyperphosphorylated andmisfolded tau occurs in soma, dendrites and axons of neurons.Neurofibrillar tangles (NFTs) are related to the aggregated tau in thesoma and are principally found in the medium-sized pyramidal neurons ofthe hippocampus, of the entorhinal cortex and of layers of theisocortex. Neuropil threads (Braak et al. 1986 Neurosci Lett., 65,351-5) are small, fragmented, tortuous processes, weaving between thecell bodies. These tortuous fibers (Duyckaerts et al. 1989 NeuropatholAppl Neurobiol., 15, 233-47) contain PHFs that accumulate in dendritesof tangle-bearing neurons (Braak and Braak 1988 Neuropathol ApplNeurobiol., 14, 39-44). Neuropil threads invariably accompanyneurofibrillary tangles in AD and appear at an early stage of theneurofibrillary degeneration (Braak et al. 1994 Acta Neuropathol., 87,554-67). Finally, another tau lesion: dystrophic neurites containinghyperphosphorylated tau are found around neuritic plaques. They aremainly constituted of axonal processes enriched in paired helicalfilaments (PHF) (Kidd 1964 Brain, 87, 307-20). Interestingly, dystrophicneurites can also be immunoreactive for Amyloid Precursor Protein (APP),mitochondrial porin and chromogranin-A (Su et al. 1998 ActaNeuropathol., 96, 463-71; Dickson et al. 1999 Exp Neurol., 156, 100-10;Woodhouse et al. 2006 Acta Neuropathol., 112, 429-37; Pérez-Gracia etal. 2008 Acta Neuropathol., 116, 261-8).

Aside from Alzheimer's disease (AD), a family of relatedneurodegenerative diseases called tauopathies is also characterized bythe deposition of tau-containing neurofibrillary tangles. These includeprogressive supranuclear palsy (PSP; Steele-Richardson-Olszewskidisease), corticobasal degeneration (CBD), Pick's disease (PD),frontotemporal dementia with parkinsonism linked to chromosome 17(FTDP-17) caused by tau mutations. In every one of these tauopathies theneurofibrillary pathology is made up of abnormally hyperphosphorylatedtau and these pathological changes in the neocortex are associated withdementia. Different tau aggregates and filaments could be present indifferent diseases, raised by different composition of tau isoforms (tau3R or 4R). For instance, in Pick's disease most of the tau is 3R isoformdue to the exclusion of exon 10 which codes for the second microtubulebinding repeat (R2). In contrast, in CBD and PSP, most of the tau is 4R(Morris et al. 1999 Mov Dis., 14, 731-6; Sergeant et al. 2005 BiochimBiophys Acta., 1739, 179-197; Yoshida 2006 Neuropathology, 26, 457-470;Liu and Gong 2008 Mol Neurodegener., 3, 8; Zhong et al. 2012 J BiolChem., 287, 20711-9; Avila et al. 2013 Aging Dis., 4, 23-8; Iqbal et al.2013 Front Neurol., 4, 112).

In vivo detection of amyloid plaques and tau lesions is critical toperform early diagnosis of Alzheimer's disease (AD) and of othertauopathies and to follow-up the effect of therapies. Current detectionof these lesions is based on molecules that have a good affinity andspecificity for these alterations. To date, in human, positron emissiontomography (PET) and magnetic resonance imaging (MRI) have enabledvisualization of amyloid plaques. However, the majority of compoundsused in in vivo imaging modalities do not bind tau lesions. Therefore,there is a need to develop specific tau imaging agents. Recently,Maruyama et al. demonstrated the possibility to visualize in vivo taulesions in AD patients using [¹¹C]-PBB3-PET (Maruyama et al. 2013Neuron., 79, 1094-108). Nevertheless, the need to radiolabel thecompound and low spatial resolution are still the main disadvantages ofPET-based methods. Conversely, MRI has a much higher spatial resolutionand is widely available for animal and patient imaging. Even so, the lowsensitivity of the MRI requires the development of dedicated contrastagents able to detect NFTs in patients.

Conventional immunoglobulins are heterotetramers composed of two heavychains and two light chains with a combined molecular weight of about150 kDa. In members of the family Camelidae a significant proportion ofserum antibodies are homodimeric IgGs with a molecular weight of about80 kD (Hamers-Casterman et al. 1993 Nature, 363, 446-448). These heavychain immunoglobulins (Ig) contain three domains and their variableregion is referred to as VHH. Recombinant VHHs (˜12-14 kD in size)constitute intact antigen-binding domains and exhibit a broadantigen-binding repertoire. Their hypervariable regions are expanded andexhibit unique characteristics, such as the substitution of three tofour hydrophobic framework residues (which interact with the V_(L) inconventional antibodies) by more hydrophilic amino acids. To stabilizethe enlarged CDRs, VHHs may possess in addition of the canonicaldisulfide bond, an extra disulfide bound between CDR1 and CDR3 indromedaries and CDR2 and CDR3 in llamas (Harmsen and De Haard 2007 ApplMicrobiol Biotechnol., 77, 13-22; Muyldermans 2001 J Biotechnol., 74,277-302). The extended CDR3 loop can adopt a convex conformation,whereas conventional paratopes are limited to concave or flat structures(Muyldermans 2001 J Biotechnol., 74, 277-302). These features allow VHHsto recognize unique epitopes that are poorly immunogenic forconventional antibodies (Lafaye et al. 2009 Mol Immuno., 46, 695-704;Wernery 2001 J Vet Med B Infect Dis Vet Public Health., 48, 561-568).Although VHHs are by definition monovalent antibodies, which by defaultexclude any avidity effect, their biological activity measured as IC₅₀in vitro can be similar to conventional, bivalent antibody molecules(Thys et al. 2010 Antiviral Res., 87, 257-264).

Methods, such as phage display, have been described to selectantigen-specific VHH from VHH repertoires of immunized camels or llamas.The VHH genes are cloned in phage display vectors, the antigen bindersare obtained by panning and selected VHH are expressed in bacteria. Therecombinant VHHs have a number of advantages compared with theconventional antibody fragments (Fab or scFv), because only one domainhas to be cloned and because these VHHs are well expressed, highlysoluble in aqueous environments and are stable at high temperature.Because of their small size of about 12-14 kDa, VHHs rapidly pass therenal filter, which has a cutoff of about 60 kDa, resulting in rapidblood clearance. In addition, the small size results in a fast tissuepenetration. The VHH short serum half-life of about 2 hours, compared to4 h for scFv and 50 h for IgG, is advantageous for in vivo diagnosisusing imaging and for the targeting of VHHs coupled to a substance ofinterest for treating a disorder, as one can expect that unspecificallybound VHH will be quickly removed from the tissues. Further, a VHHhaving an isoelectric point of at least 8.5 is able to transmigrateacross the BBB by micropinocytosis and absorptive-mediated endocytosis.Such a VHH can be used for the preparation of a peptide vector fordelivering a substance of interest across a mammal blood-brain barrier(International Applications WO 2009/004495 and WO 2010/004432).

Within the framework of research that has led to the present invention,the inventors have immunized two alpacas (Lama pacos) against aphosphorylated-tau (phospho-tau, phosphor-tau or p-tau) enrichedAlzheimer's disease brain (hippocampal) extract and phospho-tauproteins. VHHs targeting phospho-tau were identified by phage displaylibrary construction and panning. Following bacterial expression,selected VHHs were screened by immunobloting and ELISA against p-tauepitopes before being tested on brain sections from human cases withneuropathologically-confirmed AD or other tauopathies and fromtransgenic mouse model harboring tangles pathologies. The inventorsidentified a VHH, referred to as Tau-A2 or A2, that immunolabels somatictangles, neuropil threads and dystrophic neurites in mutated tautransgenic mice as well in AD human brain samples. In addition VHHTau-A2 allows detection of glial p-tau inclusions in other tauopathies,fronto-temporal dementia, corticobasal degeneration and progressivesupranuclear palsy. This VHH Tau-A2 has the amino acid sequence SEQ IDNO. 4, that comprises a CDR1 (Complementarity Determining Region 1) ofamino acid sequence SEQ ID NO. 1, a CDR2 of amino acid sequence SEQ IDNO. 2 and a CDR3 of amino acid sequence SEQ ID NO. 3. VHH Tau-A2specifically recognizes the phosphorylated serine 422 (pS422) in a C-tertau phospho-peptide. Further, labeling of VHH Tau-A2 with theparamagnetic agent gadolinium (Gd) or Alexa Fluor® 488 fluorophore wasperformed using a site-specific coupling approach, resulting infunctionally effective VHH conjugates. After intravenous administrationof fluorescent VHH Tau-A2, live two-photon microscopy showed gradualextravasation of the VHH Tau-A2 from blood vessels and penetration inbrain parenchyma with an exquisite tropism for tangles.

Accordingly, the present invention provides an isolated variable domainof a camelid heavy-chain antibody (referred to as VHH), characterized inthat it is directed against a phosphorylated tau protein, preferablydirected against the phosphorylated serine 422 of a phosphorylated tauprotein.

As used herein, a tau protein refers to the well known six isoforms oftau protein (Goedert et al. 1989 Neuron, 3, 519-26; Himmler et al. 1989Mol Cel Biol., 9, 1381-8), preferably the 4R isoform.

In a preferred embodiment, the VHH of the invention is obtainable byimmunizing a camelid with the single phospho-peptide derived from theC-terminus of a tau protein of sequence CSIDMVDS(PO₃H₂)PQLATLAD (SEQ IDNO. 6) or a phospho-tau enriched Alzheimer's disease brain (preferablyhippocampal) extract from a human or a phosphorylated tau protein suchas a phosphorylated tau protein wherein the serine 422 thereof isphosphorylated, preferably with the single phospho-peptide derived fromthe C-terminus of a tau protein of sequence CSIDMVDS(PO₃H₂)PQLATLAD (SEQID NO. 6).

A phospho-tau enriched Alzheimer's disease brain (preferablyhippocampal) extract from a human can be obtained as described inMercken et al. 1992 Acta Neuropathol., 84, 265-272.

Advantageously, the VHH of the invention is obtainable by the methodcomprising the steps of:

(a) immunizing a camelid, preferably a Lama pacos, with the singlephospho-peptide derived from the C-terminus of a tau protein of sequenceCSIDMVDS(PO₃H₂)PQLATLAD (SEQ ID NO. 6) or a phospho-tau enrichedAlzheimer's disease brain (preferably hippocampal) extract from a humanor a phosphorylated tau protein such as a phosphorylated tau proteinwherein the serine 422 thereof is phosphorylated, preferably with thesingle phospho-peptide derived from the C-terminus of a tau protein ofsequence CSIDMVDS(PO₃H₂)PQLATLAD (SEQ ID NO. 6),

(b) isolating peripheral lymphocytes of the immunized camelid, obtainingthe total RNA and synthesizing the corresponding cDNAs (methods areknown in the art; for instance see Lafaye et al. 1995 Res Immunol., 146,373-82; Erratum in: 1996, Res Immunol., 147, 61),

(c) constructing a library of cDNA fragments encoding VHH domains,

(d) transcribing the VHH domain-encoding cDNAs obtained in step (c) tomRNA using PCR, converting the mRNA to ribosome display format, andselecting the VHH domain by ribosome display, and

(e) expressing the VHH domain in a vector, for instance, a suitablevector is pET22 (Novagen, Cat. No. 69744-3) and, optionally purifyingthe expressed VHH domain.

In a preferred embodiment of said method, in step (a), the camelid isimmunized at days 0, 21 and 40 with 500 μg of the peptide of sequenceCSIDMVDS(PO₃H₂)PQLATLAD (SEQ ID NO. 6) or a phospho-tau enrichedAlzheimer's disease brain (preferably hippocampal) extract from a humanor a phosphorylated tau protein wherein the serine 422 thereof isphosphorylated. The bound camelid antibodies can be detected withpolyclonal rabbit anti-camelid IgG (for instance, see Muyldermans 1994Protein Eng., 7, 1129-35) and horseradish peroxidase-labeled goatanti-rabbit antibodies.

In another preferred embodiment of said method, in step (c), saidlibrary can be constructed by amplifying by PCR the DNA fragmentsencoding the VHH domains, and ligating the PCR products obtained into aphage vector (an example of suitable phage vector is pHEN; Hoogenboom etal. 1992 J Mol Biol., 227, 381-8).

In a particular embodiment of said step (c), the DNA fragments encodingVHH domains are amplified by PCR using the primers of sequences SEQ IDNO. 7 (named CH2FORTA4) and SEQ ID NO. 8 (named VHBACKA6), and theamplified product is subjected to a second round of PCR using either ofthe primers of sequences SEQ ID NO. 9 (named VHBACKA4) and SEQ ID NO. 10(named VHFOR36). Such a method is described in the International PCTApplication No. WO 2004/044204.

Ribosome display technology enables in vitro selection of a proteintogether with the mRNA that encodes it. A DNA library coding forparticular proteins, for instance VHH fragments, is transcribed invitro. The mRNA is purified and used for in vitro translation. As themRNA lacks a stop codon, the ribosome stalls at the end of the mRNA,giving rise to a ternary complex of mRNA, ribosome and functionalprotein (Hanes and Plückthun 1997 Proc Natl Acad Sci U.S.A., 94,4937-42). A library of these ternary complexes is tested against thepotential ligand (in the case of antibodies, against the antigen). Thebinding of the ternary complex (ribosome, mRNA, protein) to the ligandallows the recovery of the encoding mRNA that is linked to it and thatcan be transcribed into cDNA by Reverse Transcriptase-PCR (RT-PCR).Cycles of selection and recovery can be iterated both to enrich rareligand-binding molecules, and to select molecules with the bestaffinity. Methods for ribosome display selections are known in the art;see for instance Mouratou et al. 2007 Proc Natl Acad Sci U.S.A., 104,17983-8.

In a preferred embodiment of the VHH of the invention, its amino acidsequence comprises, from the N-terminus to the C-terminus, the aminoacid sequence SEQ ID NO. 1 (corresponding to the CDR1 of the VHH), theamino acid sequence SEQ ID NO. 2 (corresponding to the CDR2) and theamino acid sequence SEQ ID NO. 3 (corresponding to the CDR3).

In a more preferred embodiment, said VHH consists of the amino acidsequence:

-   -   SEQ ID NO. 4, corresponding to the full-length form of VHH        Tau-A2 or    -   SEQ ID NO. 5, corresponding to a short form of VHH Tau-A2.

As used herein, the term “isolated” refers to a VHH which has beenseparated from a component of its natural environment. In someembodiments, a VHH is purified to greater than 95% or 99% purity asdetermined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectricfocusing (IEF), capillary electrophoresis) or chromatographic (e.g., gelfiltration, ion exchange or reverse phase HPLC). For review of methodsfor assessment of antibody purity, see, e.g., Flatman et al. 2007 JChromatogr B Analyt Technol Biomed Life Sci., 848, 79-87.

As used herein, the term “VHH” refers to the variable antigen-bindingdomain from a camelid (camel, dromedary, llama, alpaca, etc.)heavy-chain antibody (See Nguyen et al. 2000 EMBO J., 19, 921-930;Muyldermans 2001 J Biotechnol., 74, 277-302 and for review Vanlandschootet al. 2011 Antiviral Res. 92, 389-407). A VHH can also be namedNanobody (Nb).

Advantageously, the VHH according to the present invention has a basicisoelectric point (pI), preferably between 8.5 and 10.5, and morepreferably between 9.5 and 10.5.

The invention encompasses natural, recombinant or synthetic VHHs asdefined above.

As used herein, the term “recombinant” refers to the use of geneticengineering methods (cloning, amplification) to produce said VHH.

As used herein, the term “synthetic” refers to the production of saidVHH by in vitro chemical and/or enzymatic synthesis.

The VHH according to the present invention can be in the form of amonomer or a homomultimer, such as a homodimer or a homotrimer.

The present invention also provides a method for obtaining a VHHdirected against a phosphorylated tau protein as defined abovecomprising a step of immunizing a camelid with the singlephospho-peptide derived from the C-terminus of a tau protein of sequenceCSIDMVDS(PO₃H₂)PQLATLAD (SEQ ID NO. 6) or a phospho-tau enrichedAlzheimer's disease brain (preferably hippocampal) extract from a humanor a phosphorylated tau protein such as a phosphorylated tau proteinwherein the serine 422 thereof is phosphorylated, preferably comprisingthe steps (a) to (e) as defined above.

The present invention also provides an isolated camelid serum,preferably an alpaca serum, comprising a VHH according to the presentinvention.

The present invention also provides an isolated variant of the VHHTau-A2 of SEQ ID NO. 5, wherein said VHH variant is directed against thephosphorylated serine 422 of a phosphorylated tau protein as definedabove, and wherein the amino acid sequence of said variant has at least95% identity, or by order of increasing preference at least 96%, 97%,98% or 99% identity, with the amino acid sequence SEQ ID NO: 5.

Unless otherwise specified, the percents of identity between twosequences which are mentioned herein are calculated from an alignment ofthe two sequences over their whole length.

In a preferred embodiment of said variant, the amino acid sequencethereof comprises, from the N-terminus to the C-terminus, the amino acidsequence SEQ ID NO. 1, the amino acid sequence SEQ ID NO. 2 and theamino acid sequence SEQ ID NO. 3.

In another preferred embodiment, said variant has the amino acidsequence SEQ ID NO. 5 having the following mutations:

-   -   the Glutamine residue (Gln, Q) at position 3 of the amino acid        sequence SEQ ID NO. 5 is substituted with an amino acid residue        selected from the group consisting of Aspartic acid (Asp, D) and        Glutamic acid (Glu, E), preferably Glu,    -   the Isoleucine residue (Ile, I) at position 52 of the amino acid        sequence SEQ ID NO. 5 is substituted with an amino acid residue        selected from the group consisting of Alanine (Ala, A) and        Glycine (Gly, G), preferably Gly,    -   the Valine residue (Val, V) at position 86 of the amino acid        sequence SEQ ID NO. 5 is substituted with an amino acid residue        selected from the group consisting of Alanine (Ala, A), Serine        (Ser, S), Threonine (Thr, T), Asparagine (Asn, N), Glutamine        (Gln, Q), Aspartic acid (Asp, D), Glutamic acid (Glu, E), Lysine        (Lys, K), Arginine (Arg, R) and Glycine (Gly, G), preferably        Gly,    -   and optionally two amino acid residues are added in N-terminal        position of the amino acid sequence SEQ ID NO. 5 and are        selected from the group consisting of the dipeptides Glutamic        acid-Valine (E-V) and Aspartic acid-Valine (D-V), preferably        D-V.

In a particular embodiment, said VHH variant consists in the amino acidsequence SEQ ID NO: 15.

In another particular embodiment, said VHH variant consists in the aminoacid sequence SEQ ID NO: 16 (this VHH variant does not comprise an addeddipeptide in N-terminal position compared to the amino acid sequence SEQID NO. 5).

The present invention also provides a VHH derivative consisting of apolypeptide comprising a VHH or a VHH variant (preferably the VHHvariant of SEQ ID NO. 15) according to the present invention, providedthat said VHH or VHH variant comprised in said polypeptide is able tobind a phosphorylated tau protein, preferably the phosphorylated serine422 of a phosphorylated tau protein.

In another particular embodiment, said VHH derivative has the formulaP-C-Z or Z-C-P, preferably P-C-Z, wherein:

-   -   P is a 100-500, preferably 100-150, amino acid peptide        comprising or consisting of a VHH or VHH variant according to        the present invention, wherein said amino acid sequence has no        accessible reduced cystein residue, and preferably has no        reduced cystein residue,    -   C is a cystein residue,    -   Z is a 1-10 amino acid spacer, preferably a 1-10 neutral or        negatively charged amino acid spacer, wherein the amino acid        residues of Z are identical or different and wherein Z does not        contain a cystein residue.

In the sense of the invention, the expression “no accessible reducedcystein residue” refers to a cystein residue which is not accessible fora conjugation step as defined according to the invention.

In a preferred embodiment, the VHH derivative has the formula P-C-Z orZ-C-P, preferably P-C-Z, wherein:

-   -   P is a 100 to 500, preferably 100-150, amino acid peptide having        no accessible reduced cystein residue, and preferably has no        reduced cystein residue,    -   C is a cystein residue,    -   Z represents

a) a 2-10 amino acid spacer, preferably a 2 amino acid spacer, whereinthe amino acid residues of Z are selected from the group consisting ofserine (S), alanine (A), valine (V) and glycine (G), and more preferablyserine (S), alanine (A) and valine (V), and wherein at least two aminoacid residues of Z are different, or

b) a 2-10 amino acid spacer, preferably a 2-10 neutral or negativelycharged amino acid spacer, wherein Z comprises the dipeptideserine-alanine (S-A) or serine-valine (S-V) and wherein Z does notcontain a cystein residue.

Advantageously, the cystein residue C is sterically accessible.

Advantageously, the amino acid residues of the amino acid spacer Z areselected from the group consisting of alanine, valine, serine, leucine,isoleucine, phenylalanine, glycine, serine, threonine, tyrosine,asparagine and glutamine, preferably alanine, valine and serine.

Advantageously, when Z is as defined in a), the amino acid spacer Zcomprises 1 or at least 1 serine, more advantageously, the amino acidspacer Z consists in serine and alanine residues only or in serine andvaline residues only.

In a preferred embodiment of this VHH derivative, the amino acid spacerZ consists of a 2 amino acid sequence, such as the amino acid sequencesS-A or S-V.

The amino acid sequence P of the VHH derivative of formula P-C-Z canhave at its C-terminus a 1-10 amino acid spacer Y, preferably a 1-10neutral or negatively charged amino acid spacer, wherein the amino acidresidues of said amino acid spacer Y are identical or different, andwherein said amino acid spacer Y does not contain a cystein residue.

The amino acid sequence P of the VHH derivative of formula Z-C-P canhave at its N-terminus a 1-10 amino acid spacer Y, preferably a 1-10neutral or negatively charged amino acid spacer, wherein the amino acidresidues of said amino acid spacer Y are identical or different, andwherein said amino acid spacer Y does not contain a cystein residue.

Advantageously, the amino acid residues of the amino acid spacer Y areselected from the group consisting of alanine, valine, serine, leucine,isoleucine, phenylalanine, glycine, serine, threonine, tyrosine,asparagine and glutamine, preferably alanine, valine, serine andglycine.

Preferably, the amino acid spacer Y represents a 4 neutral amino acidspacer, such as the amino acid sequence G-G-G-S(SEQ ID NO. 11).

The amino acid sequence P of the VHH derivative of formula P-C-Z canalso have at its N-terminus a 1-50 amino acid sequence X, wherein theamino acid residues of said amino acid sequence X are identical ordifferent, and wherein said amino acid sequence X does not contain acystein residue.

The amino acid sequence P of the VHH derivative of formula Z-C-P canalso have at its C-terminus a 1-50 amino acid sequence X, wherein theamino acid residues of said amino acid sequence X are identical ordifferent, and wherein said amino acid sequence X does not contain acystein residue.

The amino acid sequence X can comprise a tag such as a 6×His tag (SEQ IDNO. 12) and an enzyme cleavage site, such as the thrombin cleavage siteof amino acid sequence LVPRGS (SEQ ID NO. 13).

In a preferred embodiment, the VHH derivative according to the presentinvention has the formula P′-C-Z, P′-Y-C-Z, X-P′-C-Z, X-P′-Y-C-Z,Z-C-P′, Z-C-Y-P′, Z-C-P′-X, or Z-C-Y-P′-X, wherein P′ is a VHH or VHHvariant according to the present invention.

The present invention also provides an isolated oligopeptide of formulaP-C-Z or Z-C-P, preferably P-C-Z as defined above.

Advantageously, said VHH derivative comprises, from the N-terminus tothe C-terminus, an amino acid tag such as a 6×His tag, an enzymecleavage site, such as a thrombin cleavage site, a VHH or VHH variantaccording to the present invention, an amino acid spacer, a cystein anda second amino acid spacer. Such a VHH derivative corresponds to a VHHderivative of formula X-P′-Y-C-Z, wherein P′ is a VHH or VHH variant.

In a preferred embodiment, said VHH derivative has the amino acidsequence SEQ ID NO. 14 (Tau-A2-SH).

In another preferred embodiment, said VHH derivative has the amino acidsequence SEQ ID NO. 17 (Tau-A2var-SH). This VHH derivative comprises theVHH variant of SEQ ID NO. 15 according to the invention.

The present invention also provides an isolated polynucleotide encodinga VHH, or VHH variant or a VHH derivative according to the presentinvention.

A polynucleotide according to the present invention may be obtained bywell-known methods of recombinant DNA technology and/or of chemical DNAsynthesis.

The present invention also provides a recombinant expression cassettecomprising a polynucleotide according to the present invention under thecontrol of a transcriptional promoter allowing the regulation of thetranscription of said polynucleotide in a host cell. Said polynucleotidecan also be linked to appropriate control sequences allowing theregulation of its translation in a host cell.

The present invention also provides a recombinant vector (e.g., arecombinant expression vector) comprising a polynucleotide according tothe present invention. Advantageously, said recombinant vector is arecombinant expression vector comprising an expression cassetteaccording to the present invention.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

The present invention also provides a host cell containing a recombinantexpression cassette or a recombinant vector according to the presentinvention. The host cell is either a prokaryotic or eukaryotic hostcell.

The terms “host cell” refers to a cell into which exogenous nucleic acidhas been introduced, including the progeny of such cells. Host cellsinclude “transformants” and “transformed cells”, which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A prokaryotic host cell expressing VHH Tau-A2 of amino acid sequence SEQID NO. 4 with a 6×Histidine tag was deposited on Jan. 16, 2014, at theCollection Nationale de Cultures de Microorganismes (CNCM), 28 rue du DrRoux, 75724 Paris Cedex 15, France, under the number I-4835.

A prokaryotic host cell expressing VHH Tau-A2-SH of amino acid sequenceSEQ ID NO. 14 was deposited on Jan. 16, 2014, at the CollectionNationale de Cultures de Microorganismes (CNCM), 28 rue du Dr Roux,75724 Paris Cedex 15, France, under the number I-4836.

A prokaryotic host cell expressing VHH Tau-A2var-SH (also named TauA2VAR-SH) of amino acid sequence SEQ ID NO. 17 was deposited on Jan. 21,2015, at the Collection Nationale de Cultures de Microorganismes (CNCM),28 rue du Dr Roux, 75724 Paris Cedex 15, France, under the numberI-4951.

The present invention also provides a method for producing in a hostcell as defined above an oligopeptide of formula P-C-Z or Z-C-Paccording to the present invention, comprising the steps of:

-   -   providing a host cell containing a recombinant expression        cassette or a recombinant vector according to the present        invention,    -   culturing said host cell,    -   and optionally purifying the oligopeptide of formula P-C-Z or        Z-C-P.

Methods for purifying an oligopeptide are well known in the art, such aschromatography (e.g., ion exchange chromatography, gel permeationchromatography and reversed phase chromatography).

The present invention also provides a diagnostic or therapeutic agentcomprising a VHH, a VHH variant or a VHH derivative (in particular a VHHderivative of formula P-C-Z or Z-C-P as defined above) according to thepresent invention, linked, directly or indirectly, covalently ornon-covalently to a substance of interest.

The substance of interest according to the present invention may or maynot permeate the mammal or human blood-brain barrier. If the substanceof interest permeates said blood-brain barrier, then the use of a VHH, aVHH variant or a VHH derivative (in particular a VHH derivative offormula P-C-Z or Z-C-P as defined above) according to the presentinvention can allow enhancing the delivery of said substance of interestacross the blood-brain barrier.

In an embodiment, said substance of interest is a diagnostic ortherapeutic compound.

In another embodiment, said substance of interest is a liposome or apolymeric entity comprising a diagnostic or therapeutic compound(Villaraza et al. 2010 Chem Rev., 110, 2921-2959).

Advantageously, said diagnostic compound is selected from the groupconsisting of:

-   -   an enzyme such as horseradish peroxidase, alkaline phosphatase,        glucose-6-phosphatase or beta-galactosidase;    -   a fluorophore such as green fluorescent protein (GFP), blue        fluorescent dyes excited at wavelengths in the ultraviolet (UV)        part of the spectrum (e.g. AMCA        (7-amino-4-methylcoumarin-3-acetic acid); Alexa Fluor® 350),        green fluorescent dyes excited by blue light (e.g. FITC, Cy2,        Alexa Fluor® 488), red fluorescent dyes excited by green light        (e.g. rhodamines, Texas Red, Cy3, Alexa Fluor dyes 546, 564 and        594), or dyes excited with far-red light (e.g. Cy5) to be        visualized with electronic detectors (CCD cameras,        photomultipliers);    -   a radioisotope such as ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁶⁸Ga, ⁸²Rb, ⁴⁴Sc,        ⁶⁴Cu, ⁸⁶Y, ⁸⁹Zr, ¹²⁴I, ¹⁵²Tb that can be used for PET imaging or        ⁶⁷Ga, ^(81m)Kr, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁵I, ¹³³Xe, ²⁰¹Tl,        ¹⁵⁵Tb, ^(195m)Pt that can be used for SPECT/scintigraphic        studies, or ¹⁴C, ³H, ³⁵S, ³²P, ¹²⁵I that can be used for        autoradiography or in situ hybridisation, or ²¹¹At-, ²¹²Bi-,        ⁷⁵Br-, ⁷⁶Br-, ¹³¹I-, ¹¹¹In, ¹⁷⁷Lu-, ²¹²Pb-, ¹⁸⁶Re-, ¹⁸⁸Re-,        ¹⁵³Sm-, ⁹⁰Y that can be used to label the compounds;    -   a NMR or MRI contrast agent such as the paramagnetic agents        gadolinium (Gd), dysprosium (Dy) and manganese (Mn), and the        superparamagnetic agents based on iron oxide (such as MION, SPIO        or USPIO) or iron platinium (SIPP), and X-nuclei such as ¹⁸F,        ¹³C, ²³Na, ¹⁷O, ¹⁵N;    -   a nanoparticle such as gold nanoparticles (B. Van de Broek et        al., ACSNano, Vol. 5, No. 6, 4319-4328, 2011) or quantum dots        (A. Sukhanova et al., 2012 Nanomedicine, 8 516-525).

In a preferred embodiment, said diagnostic compound is a fluorophore,more preferably Alexa Fluor® 488, or a MRI contrast agent, morepreferably gadolinium.

When the diagnostic agent is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example ⁹⁹Tc or ¹²³I, ora spin label for nuclear magnetic resonance (NMR) imaging (also known asMRI), such as ¹³C, ⁹F, Fe, Gd, ¹²³I, ¹¹¹In, Mn, ¹⁵N or ⁷O.

Advantageously, said therapeutic compound is selected from a peptide, anenzyme, a nucleic acid, a virus and a chemical compound. It can be ananalgesic compound, an anti-inflammatory compound, an antidepressantcompound, an anticonvulsant compound, a cytotoxic compound or ananti-neurodegenerative compound.

The substance of interest as defined above can be directly andcovalently or non-covalently linked to the VHH, VHH variant or VHHderivative (in particular a VHH derivative of formula P-C-Z or Z-C-P asdefined above) according to the present invention either to one of theterminal ends (N or C terminus) of said VHH, VHH variant or VHHderivative (in particular a VHH derivative of formula P-C-Z or Z-C-P asdefined above), or to the side chain of one of the amino acids of saidVHH, VHH variant or VHH derivative. The substance of interest can alsobe indirectly and covalently or non-covalently linked to said VHH, VHHvariant or VHH derivative by means of a spacer either to one of theterminal ends of said VHH, VHH variant or VHH derivative (in particulara VHH derivative of formula P-C-Z or Z-C-P as defined above), or to aside chain of one of the amino acids of said VHH, VHH variant or VHHderivative. Conventional linking methods of a substance of interest to apeptide, in particular an antibody, are known in the art (e.g., SeeTemynck and Avrameas 1987 “Techniques immunoenzymatiques” Ed. INSERM,Paris; Hermanson, 2010, Bioconjugate Techniques, Academic Press).

Many chemical cross-linking methods are also known in the art.Cross-linking reagents may be homobifunctional (i.e., having twofunctional groups that undergo the same reaction) or heterobifunctional(i.e., having two different functional groups). Numerous cross-linkingreagents are commercially available. Detailed instructions for their useare readily available from the commercial suppliers. A general referenceon polypeptide cross-linking and conjugate preparation is: WONG,Chemistry of protein conjugation and cross-linking, CRC Press (1991).

The VHH, VHH variant or VHH derivative (in particular a VHH derivativeof formula P-C-Z or Z-C-P as defined above) according to the presentinvention may be labeled with specific radioisotopes or NMR or MRIcontrast agents or fluorophores or nanoparticles or enzymes usinggeneral organic chemistry techniques known in the art. See, e.g., March,J. ADVANCED ORGANIC CHEMISTRY: REACTIONS, MECHANISMS, AND STRUCTURE (3rdEdition, 1985); Hermanson, 2010, Bioconjugate Techniques, AcademicPress.

In addition, the VHH, VHH variant or VHH derivative according to thepresent invention also may be labeled with any suitable radioactiveiodine isotope, such as, but not limited to ¹³¹I, ¹²⁵I, Or ¹²³I, byiodination of a diazotized amino derivative directly via a diazoniumiodide (see Greenbaum 1936 F Am J Pharm., 108, 17), or by conversion ofthe unstable diazotized amine to the stable triazene, or by conversionof a non-radioactive halogenated precursor to a stable tri-alkyl tinderivative which then can be converted to the iodo compound by severalmethods well known to the art. See, Satyamurthy and Barrio 1983 J OrgChem., 48, 4394; Goodman et al. 1984 J Org Chem., 49, 2322; Mathis etal. 1994 J Labell Comp Radiopharm., 905; Chumpradit et al. 1991 J MedChem., 34, 877; Zhuang et al. 1994 J Med Chem., 37, 1406; Chumpradit etal. 1994 J Med Chem., 37, 4245.

In particular, the VHH, VHH variant or VHH derivative according to thepresent invention can be labeled with ¹²³I for SPECT by any of severaltechniques known to the art. See, e.g., Kulkarni 1991 Int J Rad ApplInst. (Part B) 18, 647.

The VHH, VHH variant or VHH derivative according to the presentinvention also may be radiolabeled with known metal radiolabels, such asTechnetium-99m (^(99m)Tc). Modification of the substituents to introduceligands that bind such metal ions can be effected without undueexperimentation by one of ordinary skill in the radiolabeling art. Themetal radiolabeled VHH, VHH variant or VHH derivative according to thepresent invention can then be used to detect neurofibrillary tangles,neuropil threads or dystrophic neurites. Preparing radiolabeledderivatives of ^(99m)Tc is well known in the art. See, for example,Zhuang et al. 1999 Nucl Med Biol., 26, 217-24; Oya et al. 1998 Nucl MedBiol., 25, 135-40; Horn et al. 1997 Nucl Med Biol., 24, 485-98.

The invention also relates to coupling methods for obtaining a VHH, VHHvariant or VHH derivative (in particular a VHH derivative of formulaP-C-Z or Z-C-P as defined above) according to the invention coupled,directly or indirectly, with a substance of interest (functionalconjugate).

According to a first strategy, a VHH, VHH variant or VHH derivativeaccording to the invention is conjugated to a substance of interest byusing a non-site specific approach. Said non-site specific methodcomprises a conjugation step of a substance of interest with a VHH, VHHvariant or VHH derivative according to the invention.

When the substance of interest is a metal, such as a NMR or MRI contrastagent (for example, paramagnetic agents gadolinium (Gd), dysprosium (Dy)and manganese (Mn), and superparamagnetic agents based on iron oxide oriron platinium, and X-nuclei such as ¹⁸F, ¹³C, ²³Na, ¹⁷O, ¹⁵N, or suchas a metallic radioisotope (for example, ⁹⁰Y, ¹⁷⁷Lu, ⁶⁴Cu, ^(99m)Tc,¹¹¹In, ²¹²Pb, ²¹²Bi), the non-site specific method implements achelating agent and comprises the following steps:

(i) the conjugation of a chelating agent activated in the form of anester or an anhydride, preferably in the form of an ester, with lysineresidues of VHH, VHH variant or VHH derivative according to theinvention, and

(ii) the chelation of the ligand of step (i) with a substance ofinterest.

An alternative of the non-site specific method implementing a chelatingagent is a method in which the substance of interest is “pre-chelated”with a chelating agent, such method comprising the following steps:

(i′) the chelation of the substance of interest with a chelating agentactivated in the form of an ester or an anhydride, preferably in theform of an ester, and

(ii′) the conjugation of the pre-chelated substance of interest of step(i′) with lysine residues of VHH, VHH variant or VHH derivativeaccording to the invention.

During the conjugation step (i) or (ii′), the temperature may vary from1 to 40° C., and preferably from 4 to 20° C. The solution may be stirredfrom 1 to 6 hours. Preferably, the pH is maintained between 7 and 8.5during the conjugation step (i) or (ii′).

The conjugation step (i) or (ii′) can be performed in PBS/NaCl with orwithout imidazole.

During the conjugation step (i) or (ii′), the chelating agent activatedin the form of an ester or an anhydride may be dissolved in a buffersolution, such as a phosphate buffered saline (PBS) solution.

In a preferred embodiment, the molar ratio between the chelating agentactivated in the form of an ester or an anhydride and the aminofunctions of the lysine residues of VHH, VHH variant or VHH derivativeranges from 1 to 10, and is preferably of 4.

Between the conjugation step (i) and the chelation step (ii), or betweenthe chelation step (i′) and the conjugation step (ii′), there may have abuffer exchange step by diafiltration or dialyse. Advantageously, thesolution is diafiltrated, for example with a Vivaspin™ device. Duringthis buffer exchange step, the medium is cooled at a temperature rangingfrom 1 to 5° C. During this buffer exchange step, the buffer solution isexchanged for example with a sodium acetate solution, preferably understirring from 0 to 6 hours, and more preferably from 2 to 3 hours.

During the chelation step (ii) or (i′), the solution is stirred from 1to 4 hours, preferably from 2 to 3 hours. The chelation step ispreferably performed from 1 to 60° C., and more preferably at 4° C.

Then, there may have a second buffer exchange step by diafiltration ordialyse. Advantageously, the solution is diafiltrated, for example witha Vivaspin™ device. During this second buffer exchange step, the mediumis cooled at a temperature ranging from 1 to 5° C. During this seconddiafiltration step, the buffer solution is exchanged for example with amixture of PBS containing NaCl (PBS/NaCl; advantageously 300 mM NaCl),and may be concentrated by the same method (diafiltration).

Depending on the number of lysine, the substance of interest averagedensity per VHH, VHH variant or VHH derivative may vary between 0 andthe number of lysine+1. Preferably, the substance of interest averagedensity per VHH, VHH variant or VHH derivative may vary between 0 and 6.

According to a second strategy, a VHH derivative of formula P-C-Z orZ-C-P according to the invention is conjugated to a substance ofinterest by using a site specific approach. The site specific approachhas the following advantages:

-   -   the labeled VHH derivative of formula P-C-Z or Z-C-P is        chemically-defined as this method affords well-defined        conjugates which is an essential feature in the perspective of        human use (quality control, safety . . . ),    -   the method is easy and standard as the VHH derivative of formula        P-C-Z or Z-C-P labeling with the substance of interest can be        performed in a single step with short reaction time and        straightforward procedure. There is no need for in-process        monitoring and no trade-off to achieve between the labeling        degree and the binding properties. These are key advantages for        further optimization, experiment repeatability, and production        scale-up,    -   the method does not affect VHH derivative of formula P-C-Z or        Z-C-P key properties: for instance, the pI of the conjugate is        maintained above 8.5 which should allow for the BBB crossing.        Furthermore, there is no remaining unlabeled VHH derivative of        formula P-C-Z or Z-C-P which may compete with the conjugate for        the target; the mild conditions with short reaction time at        physiological pH prevent the VHH derivative of formula P-C-Z or        Z-C-P from potential degradation and/or loss of activity,    -   the method is versatile as it allows a flexible and modular        approach where various VHH derivatives of formula P-C-Z or Z-C-P        and contrast agents, fluorophores or other molecules of        interest, can be prepared separately, and then combined in a        single step. As a result, a set of conjugates are easily        accessible for optimization and downstream evaluation by IHC        (immunohistochemistry) and MRI (magnetic resonance imaging), and        above all    -   the method allows an improvement of the overall yield whilst        reducing the number of steps reaction, without side reactions on        the lysine or the histidine of the VHH, and with an overall        maintenance of the function and the 3D structure of the VHH.

The site specific method according to the invention comprises aconjugation step of a VHH derivative of formula P-C-Z or Z-C-P accordingto the invention by thio-addition (conjugation step) with athiol-reactive compound bearing a substance of interest, such as amaleimido compound of formula (I) or (I′) as defined below bearing asubstance of interest. Whereas the non-site specific conjugationrequired an initial buffer exchange, the site-specific conjugationbetween the VHH, especially Tau-A2-SH or Tau-A2var-SH, and thethiol-reactive compound bearing a substance of interest can beimplemented directly in a PBS/NaCl/imidazole buffer. Specificthio-addition on cystein could be efficiently controlled in mildconditions, said strategy allowing a reduction of the number of stepreaction and an improvement of the overall yield of the process, withoutany of the potential side reactions previously mentioned in A. Papini etal., Int. J. Pept. Protein Res., 1992, 39, 348-355; B. Rudolf et al., J.Organomet. Chem, 1996, 522, 313-315; J. Paulech et al., Biochim.Biophys. Acta, 2013, 1834, 372-379.

Recombinant proteins are routinely expressed with a His-Tag which allowstheir purification by immobilized metal affinity chromatography (IMAC).When using a Ni²⁺ nitrilotriacetic acid resin, they are typically elutedin a PBS buffer containing 500 mM imidazole. In the non-site specificapproach, the nitrogens of the imidazole can promote the NHS esterhydrolysis (i.e. degrade the reactive species), and thereby interferewith the conjugation (G. T. Hermanson, Bioconjugate Techniques, AcademicPress, 2013; P. Cuatrecasas et al., Biochemistry, 1972, 11, 2291-2299).A buffer exchange step must therefore be included in the process betweenthe upstream affinity purification and the conjugation to remove theimidazole. Side-reaction between imidazole and maleimide groups areexpected as previously reported by several groups showing the histidineside-chain alkylation (A. Papini et al.; B. Rudolf et al.; J. Paulech etal.). Nonetheless, the thiol-reactive compound bearing a substance ofinterest could be directly conjugated to the VHH, especially Tau-A2-SHor Tau-A2var-SH, in the affinity column elution buffer, with limitedexcess of maleimide reagent and despite a large molar excess ofimidazole.

The thio-addition between the cystein of the VHH derivative of formulaP-C-Z or Z-C-P and the thiol-reactive compound, such as the maleimidocompound of formula (I) or (I′) below, can be performed at a temperatureranging from 0 to 20° C., preferably 4° C., for instance from 2 to 4hours.

The thio-addition between the cystein of the oligopeptide and thethiol-reactive compound such as the maleimido compound of formula (I) or(I′) below is preferably realized at a pH ranging from 4 to 7.5, andmore preferably at 6.8. Below pH=4, the reaction is not optimal, andabove 7.5 the reaction is non specific (reaction on lysine). Theconjugation step can be performed in PBS/NaCl with or without imidazole,and preferably in presence of imidazole.

Then, there may have a buffer exchange step by diafiltration or dialyse.Advantageously, the solution is diafiltrated, for example with aVivaspin™ device. Then, the solution may be concentrated by the samemethod (diafiltration).

However, when the conjugation step is performed in PBS/NaCl withimidazole, it is preferable not to perform subsequent diafiltration ordialyse step (in order not to remove the imidazole).

Whether it is for the non-specific method or for the specific method,the substance of interest may be as defined above.

According to a preferred embodiment, the substance of interest is atherapeutic or diagnostic compound as defined above, preferably adiagnostic compound selected from the group consisting of fluorophore,radioisotope and NMR or MRI contrast agent as defined above.

The Inventors have observed that when the substance of interest is afluorophore, or a NMR or MRI contrast agent, the synthesized conjugatesretain the critical functional properties of the unlabelled VHH.

According to a preferred embodiment, the substance of interest isfluorophore, such as a green fluorescent dyes excited by blue light, inparticular FITC, Cy2, Alexa Fluor® 488, preferably Alexa Fluor® 488.

According to another preferred embodiment, the substance of interest isa NMR or MRI contrast agent, such the paramagnetic agents gadolinium(Gd), dysprosium (Dy) and manganese (Mn), and the superparamagneticagents based on iron oxides (such as MION, SPIO or USPIO) or ironplatinium (SIPP), and X-nuclei such as ¹⁸F, ¹³C, ²³Na, ¹⁷O, ¹⁵N, andmore preferably the substance of interest is a NMR or MRI contrast agentselected from the paramagnetic agents gadolinium (Gd), dysprosium (Dy)and manganese (Mn), preferably gadolinium (Gd).

The chelating agent may be chosen among1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA),diethylene triamine penta-acetic acid (DTPA),1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A),nitrilotriacetic acid (NTA) (Chong et al. 2008 Bioconjug Chem., 19,1439-47), D-penicillamine (Pen), 2,3-dimercaptosuccinic acid (DMSA),2,3-dimercapto-1-propanesulfonic acid (DMPS) (Andersen 1999 Chem Rev.,99, 2683-2710), 2,3-dimercaptopropanol (BAL), triethylenetetramine(Trien), the ammonium tetrathiomolybdate (TTM) anion (Brewer and Askari2005 J Hepatol., 42, S13-S21), ethylenediaminetetraacetic acid (EDTA),2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid(IB4M) (Nwe et al. 2011 J Inorg Biochem., 105, 722-7), hydroxypyridinone(HOPO) (Villaraza et al., 2010 Chem Rev., 110, 2921-59).

When the substance of interest is gadolinium, DOTA is the preferredchelating agent.

The present invention also provides a VHH derivative of formula P-C-Z orZ-C-P as defined above wherein said cystein residue C is linked to atleast one substance of interest through a sulphide bond, preferablythrough a thioether or disulfide bond. Advantageously, said cysteinresidue C is linked to at least one substance of interest through athiol-reactive compound bearing said substance of interest.

In the sense of the invention, a thiol-reactive compound is a maleimido,a haloacetyl, an alkyl halide or an aziridine compound, an acryloylderivative, an arylating agent, or a thiol-disulfide exchange reagent(Hermanson G. T., 2010, Bioconjugate Techniques, Academic Press).

In the sense of the invention, a maleimido compound is a compoundbearing at least one maleimide function, preferably from 1 to 6maleimide functions, and more preferably one maleimide function.

Preferably, the thiol-reactive compound is a maleimido compound reactingwith the cystein residue C through the C—C double bond of the maleimidefunction.

The maleimido compound of the invention may be of formula (I) asfollows:

wherein:

-   -   B, B′₁, B′₂ and B″, identical or different, are independently        single bonds or spacers selected from polyols, such as        polyethylene glycol (PEG) preferably having 2 to 12 oxyethylene        (OE) units, polyolefins preferably having 2 to 12 aromatic        rings, polyalkyls preferably having 2 to 12 carbon atoms, vinyl        polymers such as poly(alkyl methacrylate) preferably having 2 to        12 methacrylate groups, polyaldehydes preferably having 2 to 12        carbonyl groups, polyacid esters preferably having 2 to 12 ester        groups,    -   D, D′ and D″, identical or different, are independently selected        from amine, amide, amino-alcohol, urea, thiourea, carbamate,        carbonate, ester, ether, thioether, aryl, heteroaryl such as        triazole, oxime groups,    -   A is a single bond or a chelating agent,    -   SI is a substance of interest,    -   X′ is an acid, amine, amide, ester, ether, alkyl, alkenyl,        alkynyl, aryl or heteroaryl function, and    -   n=1 to 100, and preferably n=1, 2 or 3.

In the sense of the present invention:

-   -   Alkyl groups are chosen among (C₁-C₁₂)alkyl groups, and        preferably (C₁-C₆)alkyl groups such as methyl, ethyl, n-propyl,        isopropyl, n-butyl, sec-butyl, tert-butyl and isobutyl radicals;    -   Alkenyl groups are chosen among hydrocarbon chains of 2 to 12        carbon atoms, preferably 2 to 6, having at least one        carbon-carbon double bond. Examples of alkenyl groups include        ethenyl, propenyl, isopropenyl, 2,4-pentadienyl;    -   Alkynyl groups are chosen among hydrocarbon chains of 2 to 12        carbon atoms, preferably 2 to 6, having at least one        carbon-carbon triple bond;    -   Aryl groups means any functional group or substituent derived        from at least one simple aromatic ring; an aromatic ring        corresponding to any planar cyclic compound having a delocalized        π system in which each atom of the ring comprises a p-orbital,        said p-orbitals overlapping themselves. More specifically, the        term aryl includes, but is not limited to, phenyl, biphenyl,        1-naphthyl, 2-naphthyl, anthracyl, pyrenyl, and the substituted        forms thereof. The aryl groups of the invention comprise        preferably 4 to 12 carbon atoms, and more preferably 5 or 6        carbon atoms;    -   Heteroaryl groups means any functional group or substituent        derived from at least one aromatic ring as defined above and        containing at least one heteroatom selected from P, S, O and N.        The term heteroaryl includes, but is not limited to, furan,        pyridine, pyrrole, thiophene, imidazole, pyrazole, oxazole,        isoxazole, triazole, thiazole, isothiazole, tetrazole,        pyridazole, pyridine, pyrazine, pyrimidine, pyridazine,        benzofurane, isobenzofurane, indole, isoindole, benzothiophene,        benzo[c]thiophene, benzimidazole, indazole, benzoxazole,        benzisoxazole, benzothiazole, quinoline, isoquinoline,        quinoxaline, quinazoline, cinnoline, purine and acridine. The        aryl and heteroaryl groups of the invention comprise preferably        4 to 12 carbon atoms, and more preferably 5 or 6 carbon atoms;        The acid, amine, amide, ester, ether and thioether groups        according to the invention have preferably 1 to 12, and more        preferably 1 to 6 carbon atoms.

According to a preferred embodiment, A is a chelating agent and thesubstance of interest SI is a fluorophore (e.g., Alexa Fluor® 488) or aNMR or MRI contrast agent (e.g., gadolinium).

Advantageously, the chelating agent A is selected from1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA),diethylene triamine pentaacetic acid (DTPA),1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacethylamide (DO3A),nitrilotriacetic acid (NTA), D-penicillamine (Pen),2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonicacid (DMPS), 2,3-dimercaptopropanol (BAL), triethylenetetramine (Trien),the ammonium tetrathiomolybdate (TTM) anion, ethylenediaminetetraaceticacid (EDTA),2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid(IB4M) or hydroxypyridinone (HOPO).

Advantageously, the substance of interest SI is gadolinium, and thechelating agent is DOTA.

According to a particularly preferred embodiment, the maleimido compoundof the invention may be of formula (I′):

wherein B, B′₁, B′₂, B″, A, SI and n are as defined above.

The maleimido compound of formula (I) or (I′) may be synthesized througha solid-phase method, preferably using Fmoc chemistry, and morepreferably on a Fmoc-Gly-Wang resin.

According to a preferred embodiment, the maleimido compound of theinvention may be of formula (I′):

wherein B, B′₁, B′₂, B″, A, SI and n are as defined above is also partof the invention.

Another object of the invention is a VHH derivative of formula P-C-Z orZ-C-P as defined above with a cystein residue linked to at least onesubstance of interest (e.g., a fluorophore such as Alexa Fluor® 488 or aNMR or MRI contrast agent such as gadolinium), and preferably linked toat least one substance of interest through a thiol-reactive compound,and more preferably a maleimido compound as defined according to theinvention, said VHH derivative of formula P-C-Z or Z-C-P beingobtainable according to the site specific method of the invention.

The present invention also provides a VHH or VHH variant conjugated to asubstance of interest obtainable according to the non-site specificmethod of the invention, and also a VHH derivative of formula P-C-Z orZ-C-P conjugated to a thiol-reactive compound such as a maleimidocompound of formula (I) bearing a substance of interest obtainableaccording to the site specific method of the invention.

If the substance of interest is a peptide, then the VHH, VHH variant orVHH derivative according to the present invention and said substance ofinterest can be produced by genetic engineering as a fusion polypeptidethat includes the VHH, VHH variant or VHH derivative according to theinvention and the suitable peptide. This fusion polypeptide canconveniently be expressed in known suitable host cells.

The VHH, the VHH variant, the VHH derivative, the therapeutic ordiagnostic agent, according to the present invention can be administeredto a subject (a mammal or a human) by injection, such as intravenous,intraarterial, intrathecally (via the spinal fluid), intraperitoneal,intramuscular or subcutaneous injection, or by intranasal instillation.

When the VHH, VHH variant or VHH derivative according to the presentinvention is administered to a human subject, then it can be humanizedin order to reduce immunogenicity in human. Methods for producinghumanized antibodies or fragments thereof are known in the art (Vinckeet al. 2009, J Biol Chem., 284, 3273-84).

A diagnostic agent according to the present invention can be used inbrain imaging, in diagnosing or monitoring a disorder mediated byneurofibrillary tangles, neuropil threads or dystrophic neurites, suchas tauopathies, including Alzheimer's disease (AD), Pick disease (PD),fronto-temporal dementia (FTD), corticobasal degeneration (CBD) andprogressive supranuclear palsy (PSP), preferably AD, FTD, CBD and PSP.

The present invention also provides a kit comprising a VHH, a VHHvariant or a VHH derivative (in particular a VHH derivative of formulaP-C-Z or Z-C-P) according to the present invention and a substance ofinterest as defined above, and optionally a diagnostic reagent.

The present invention also provides a kit comprising a diagnostic agentaccording to the present invention and a diagnostic reagent.

The kits according to the present invention can be used for brainimaging, or for diagnosing or monitoring a disorder mediated byneurofibrillary tangles, neuropil threads or dystrophic neurites, suchas tauopathies, including Alzheimer's disease (AD), Pick disease (PD),fronto-temporal dementia (FTD), corticobasal degeneration (CBD) andprogressive supranuclear palsy (PSP), preferably AD, FTD, CBD and PSP.

The present invention also provides the use of a diagnostic agentaccording to the present invention for diagnosing or monitoring adisorder mediated by neurofibrillary tangles, neuropil threads ordystrophic neurites, such as tauopathies, including Alzheimer's disease(AD), Pick disease (PD), fronto-temporal dementia (FTD), corticobasaldegeneration (CBD) and progressive supranuclear palsy (PSP), preferablyAD, FTD, CBD and PSP, in a subject.

As used herein, a “subject” is a mammal, preferably a human, and mostpreferably a human suspected of having a disorder mediated byneurofibrillary tangles, neuropil threads or dystrophic neurites, suchas tauopathies, including Alzheimer's disease (AD), Pick disease (PD),fronto-temporal dementia (FTD), corticobasal degeneration (CBD) andprogressive supranuclear palsy (PSP), preferably AD, FTD, CBD and PSP.

The present invention also provides an in vitro or ex vivo method fordiagnosing a disorder mediated by neurofibrillary tangles, neuropilthreads or dystrophic neurites, such as tauopathies, includingAlzheimer's disease (AD), Pick disease (PD), fronto-temporal dementia(FTD), corticobasal degeneration (CBD) and progressive supranuclearpalsy (PSP), preferably AD, FTD, CBD and PSP, in a subject, comprisingthe steps of:

a) contacting in vitro an appropriate biological sample from saidsubject with a diagnostic agent according to the present invention, and

b) determining the presence or the absence of phosphorylated-tau proteinin said biological sample,

the presence of said phosphorylated-tau protein indicating that saidsubject has a disorder mediated by neurofibrillary tangles, neuropilthreads or dystrophic neurites, such as tauopathies, includingAlzheimer's disease (AD), Pick disease (PD), fronto-temporal dementia(FTD), corticobasal degeneration (CBD) and progressive supranuclearpalsy (PSP), preferably AD, FTD, CBD and PSP.

Step b) can be carried out by determining the presence or the absence ofthe VHH-antigen or VHH variant-antigen complex (i.e., VHH directed to aphosphorylated-tau protein).

The present invention also provides an in vitro or ex vivo method formonitoring the progression or regression of a disorder mediated byneurofibrillary tangles, neuropil threads or dystrophic neurites, suchas tauopathies, including Alzheimer's disease (AD), Pick disease (PD),fronto-temporal dementia (FTD), corticobasal degeneration (CBD) andprogressive supranuclear palsy (PSP), preferably AD, FTD, CBD and PSP,in a subject, comprising the steps of:

a) contacting in vitro an appropriate biological sample from saidsubject with a diagnostic agent according to the present invention,

b) determining the amount of phosphorylated-tau protein in saidbiological sample, and

c) comparing the amount determined in step (b) with the amount ofphosphorylated-tau protein previously obtained for said subject,

a significant increase in amount of phosphorylated-tau proteinconstituting a marker of the progression of said disorder mediated byneurofibrillary tangles, neuropil threads or dystrophic neurites, and asignificant decrease of phosphorylated-tau protein constituting a markerof the regression of said disorder mediated by neurofibrillary tangles,neuropil threads or dystrophic neurites.

As used herein the terms “significant increase” and “significantdecrease” refer to a higher amount or lower amount respectively ofphosphorylated-tau protein in an appropriate biological sample withrespect to the amount of phosphorylated-tau protein in an appropriatebiological sample from said subject, that was previously determined andused as a reference amount.

Step b) can also be carried out by determining the amount of theVHH-antigen or VHH variant-antigen complex.

Said appropriate biological sample can be a brain biopsy or post-mortembrain tissue.

According to the aspect of the invention which relates to a method ofdetecting neurofibrillary tangles, neuropil threads or dystrophicneurites in brain biopsy or post-mortem brain tissue, the method mayinvolve incubating formalin-fixed tissue with a solution of a diagnosticagent according to the invention. Upon incubation, the diagnosticcompound labels the neurofibrillary tangles, neuropil threads ordystrophic neurites in the tissue, and the stained or labeledneurofibrillary tangles, neuropil threads or dystrophic neurites can bedetected or visualized by any standard method. Such detection meansinclude microscopic techniques such as bright-field, fluorescence,laser-confocal and cross-polarization microscopy. The method ofquantifying the amount of neurofibrillary tangles, neuropil threads ordystrophic neurites in biopsy or post-mortem tissue involves, forexample incubating a diagnostic agent according to the presentinvention, or a water-soluble, non-toxic salt thereof, with homogenateof biopsy or post-mortem tissue. The tissue is obtained and homogenizedby methods well known in the art. Advantageously the diagnostic compoundis a radioisotope-labeled compound, although other diagnostic compoundssuch as enzymes, fluorophores, nanoparticles or NMR or MRI contrastagents can be used.

The present invention also provides a method for in vivo imagingneurofibrillary tangles, neuropil threads or dystrophic neurites in asubject comprising the steps of:

a) administrating a detectable quantity of a diagnostic agent accordingto the present invention in a subject, preferably a human and,

b) detecting the diagnostic agent in said subject by an imaging method.

This method according to the present invention allows determining thepresence and location of neurofibrillary tangles, neuropil threads ordystrophic neurites in brain of a subject, preferably a human.

As used herein a “detectable quantity” means that the amount of thediagnostic agent that is administered is sufficient to enable detectionof binding of the diagnostic agent to phosphorylated-tau protein.

As used herein an “imaging effective quantity” means that the amount ofthe diagnostic agent that is administered is sufficient to enableimaging of binding of said diagnostic agent to phosphorylated-tauprotein.

Imaging methods include non-invasive neuroimaging techniques such asmagnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imagingsuch as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT), used to detect neurofibrillary tangles,neuropil threads or dystrophic neurites in vivo.

For purposes of in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given label. For instance,gadolinium, iron or manganese based contrast agents can be used todetect the VHH, VHH variant or VHH derivative according to the presentinvention linked to said substances of interest by magnetic resonancespectroscopy (MRS) or imaging (MRI). Radioactive isotopes such as ¹⁹F,fluorophores such as Alexa Fluor® 488 or NMR or MRI contrast agent suchas gadolinium are also particularly suitable for in vivo imaging in themethods of the present invention. The type of instrument used will guidethe selection of the substances of interest. For instance, theradionucleide chosen must have a type of decay detectable by a giventype of instrument. Another consideration relates to the half-life ofthe contrast agent or radionuclide. For radioisotopes, the half-lifeshould be long enough so that it is still detectable at the time ofmaximum uptake by the brain, but short enough so that the subject doesnot sustain deleterious radiation. The radiolabeled VHH, VHH variant orVHH derivative according to the present invention can be detected usinggamma imaging wherein emitted gamma irradiation of the appropriatewavelength is detected. Methods of gamma imaging include, but are notlimited to, SPECT and PET. Preferably, for SPECT detection, the chosenradioisotope will lack a particulate emission, but will produce a largenumber of photons in a 140-200 keV range. For PET detection, theradiolabel will be a positron-emitting radionuclide such as ¹⁹F whichwill annihilate to form two 511 keV gamma rays which will be detected bythe PET camera.

Generally, the dosage of the detectable diagnostic agent will varydepending on considerations such as age, condition, sex, and extent ofdisorder in the patient, contraindications, if any, concomitanttherapies and other variables, to be adjusted by a physician skilled inthe art. Administration to the subject may be local or systemic andaccomplished intravenously, intraarterially, intrathecally (via thespinal fluid) or the like. Administration may also be intradermal orintracavitary, depending upon the body site under examination. After asufficient time has elapsed for the compound to bind with thephosphorylated tau protein, for example 30 minutes to 48 hours, the areaof the subject under investigation is examined by routine imagingtechniques such as MRS/MRI, SPECT, planar scintillation imaging, PET,and any emerging imaging techniques, as well. The exact protocol willnecessarily vary depending upon factors specific to the patient, asnoted above, and depending upon the body site under examination, methodof administration and type of label used; the determination of specificprocedures would be routine to the skilled artisan.

The present invention also provides a VHH, VHH variant or a VHHderivative according to the invention, in particular a VHH derivative offormula P-C-Z or Z-C-P, linked to a diagnostic compound according to thepresent invention as a diagnostic agent.

The present invention also provides a pharmaceutical compositioncomprising a therapeutic agent as defined above and a pharmaceuticallyacceptable carrier.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, Ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes, cationic lipids and non-aqueous vehicles such asfixed oils may also be used. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with atherapeutic agent as defined hereabove, use thereof in the compositionof the present invention is contemplated.

The present invention also provides a VHH, a VHH variant, a VHHderivative, a therapeutic agent or a pharmaceutical compositionaccording to the present invention as a medicament, in particular foruse in the treatment of a disorder mediated by neurofibrillary tangles,neuropil threads or dystrophic neurites, such as tauopathies, includingAlzheimer's disease (AD), Pick disease (PD), fronto-temporal dementia(FTD), corticobasal degeneration (CBD) and progressive supranuclearpalsy (PSP), preferably AD, FTD, CBD and PSP.

The present invention also provides a method for preventing or treatinga disorder mediated by neurofibrillary tangles, neuropil threads ordystrophic neurites, such as tauopathies, including Alzheimer's disease(AD), Pick disease (PD), fronto-temporal dementia (FTD), corticobasaldegeneration (CBD) and progressive supranuclear palsy (PSP), preferablyAD, FTD, CBD and PSP, comprising administering to a subject in needthereof a therapeutic agent or a pharmaceutical composition according tothe present invention.

As used herein, the terms “treatment” or “treating” includes theadministration of the VHH, the VHH variant, the VHH derivative, thetherapeutic agent or the pharmaceutical composition according to thepresent invention to a patient who has a disorder, a symptom of disorderor a predisposition toward a disorder, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisorder, the symptoms of the disorder, or the predisposition towarddisorder.

The term “preventing” means that the progression of a disorder mediatedby neurofibrillary tangles, neuropil threads or dystrophic neurites,such as tauopathies, including Alzheimer's disease (AD), Pick disease(PD), fronto-temporal dementia (FTD), corticobasal degeneration (CBD)and progressive supranuclear palsy (PSP), preferably AD, FTD, CBD andPSP, is reduced and/or eliminated, or that the onset of a disordermediated by neurofibrillary tangles, neuropil threads or dystrophicneurites, such as tauopathies, including Alzheimer's disease (AD), Pickdisease (PD), fronto-temporal dementia (FTD), corticobasal degeneration(CBD) and progressive supranuclear palsy (PSP), preferably AD, FTD, CBDand PSP, is delayed or eliminated.

In another aspect, the present invention relates to the use of a VHH, aVHH variant or a VHH derivative according to the invention, inparticular a VHH derivative of formula P-C-Z or Z-C-P, for thepreparation of a peptide vector for delivering a substance of interestas defined above across a mammal blood-brain, preferably a humanblood-brain barrier.

The present invention also provides a VHH derivative of formula P-C-Z orZ-C-P linked to a therapeutic compound according to the presentinvention as a therapeutic agent.

In addition to the preceding features, the invention further comprisesother features which will emerge from the following description, whichrefers to examples illustrating the present invention, as well as to theappended figures.

FIG. 1 shows the binding of Inti and Rascar sera against phospho-tau andtau proteins. The alpaca polyclonal antibodies were detected with rabbitanti-alpaca antibodies.

FIG. 2 shows the binding of purified VHH Tau-A2 to phospho-tau, tau(unphosphorylated) and Ova-pS422 peptide.

FIG. 3 shows the immunohistochemical staining of NFTs using the VHHTau-A2 and mAb AT8 on human paraffin sections from cases with varioustauopathies: AD (FIG. 3A), FTD (FIG. 3B), PSP (FIG. 3B) and PD (FIG.3C).

FIG. 4 shows the immunohistochemical staining of NFTs using the VHHTau-A2 and mAb AT8 on Tg4510 mouse free-floating sections.

FIG. 5 shows a western blot on AD brain extracts and phospho-tau (p-tau)revealed by VHH Tau-A2 and Tau-pS422 mAb. Lane 1 and 3: p-tau+Tau-A2 andTau-pS422 mAb, respectively. Lane 2 and 4: AD extracts+Tau-A2 andTau-pS422 mAb, respectively.

FIG. 6 shows the solid-phase synthesis of maleimido-(DOTA/Gd)₃ (compound1).

FIG. 7 shows the biochemical and histological characterization of Tau-A2and its derivatives. A: Protein profile analysis of VHH Tau-A2-SH(lane 1) and Tau-A2-S-AF488 (lane 2) by instantBlue stained SDS-PAGEgel. B: Determination of pI of Tau-A2 derivatives by NEPHGE on 3-10 IEFgel. C: In vitro imaging of NFTs by Tau-A2-S-AF488 on Tg4510 paraffinsections. The presence of VHH was revealed by the addition of a rabbitpolyclonal antibodies directed against VHH. D: Negative control: IHC onTauPS2APP VHH free paraffin section using rabbit anti-VHH Tau-A2 only.E: Direct fluorescent staining of NFTs by Tau-A2-S-AF488 in the cortexon Tg4510 free floating sections. F: Direct fluorescent staining of NFTsby Tau-A2-S-AF488 in the hippocampus on Tg4510 free floating sections.

FIG. 8 shows the in vivo imaging of NFTs after cerebral injection ofTau-A2-S-AF488 in a 8-month-old Tg4510 mouse. Arrows indicate labelingof NFTs. Scale bar=50 m.

FIG. 9 shows the in vivo two-photon imaging of Tau-A2-S-AF488 diffusingfrom blood vessels and labeling NFTs. A: Maximum intensity projection offluorescence in a cortical area of a Tg4510 mouse 1 min, 120 min, 180min after intravenous injection of Tau-A2-S-AF488 at 10 mg/kg. Whereasonly faint non-specific signal could be detected before iv injection, astrong staining of arborescent vessels was observed few second after ivinjection (Ti). Specific labeling of NFTs (white arrows) was observedafter 120 min, with a maximum intensity after 180 min (3D reconstruction(MIP)). Experiments were performed on 2 mice. B: Four hours after IVinjection of Tau-A2-S-AF488, the brain was harvested and IHC wasperformed on 5 μm-paraffin sections. Rabbit polyclonal anti-VHHantibodies were used to reveal the presence and the labeling of NFTs byTau-A2-S-AF488.

FIG. 10 shows the optimization of the amino acid sequence of Tau-A2-SHfor expression. Tau-A2-SH of SEQ ID NO. 14. Tau-A2var-SH of SEQ ID NO.17.

FIG. 11 shows the biochemical and histological characteristics of Tau-A2variant. A: Protein profile analysis of VHH Tau-A2-SH (left) andTau-A2var-SH (right) by instant Blue stained SDS-PAGE gel. B:Determination of pI of Tau-A2-SH (lane 1) and Tau-A2var-SH (lane 2) byNEPHGE on 3-10 IEF gel. C: ELISA Binding of VHH Tau-A2-SH and VHHTau-A2var-SH on coated Phospho-Tau protein. A negative control wasperformed by using an irrelevant VHH. The VHH binding was revealed bythe addition of a mouse anti-His tag mAb revealed by an anti-mouseantibody labelled with peroxydase D: Comparison of VHH Tau-A2-SH and VHHTau-A2var-SH by IHC on Tg4510 mice. VHH were used at the concentrationof 2 μg/ml and their presence was revealed by anti-His mouse mAbfollowed by a peroxydase labelled anti-mouse-Ig.

FIG. 12 shows the in vivo imaging of NFTs after intravenous injection ofTau-A2var-S-(DOTA/Gd)₃ in a 8-month-old Tg4510 mouse. Four hours afterIV injection of Tau-A2var-S-(DOTA/Gd)₃, the brain was harvested and IHCwas performed on 5 μm-paraffin sections. Rabbit polyclonal anti-VHHantibodies were used to reveal the presence and the labeling of NFTs byTau-A2var-S-(DOTA/Gd)₃.

EXAMPLE I Generation of Anti-Phosphorylated Tau VHHS Coupled to AlexaFluor® 488 or Gadolinium Contrast Agent and their Evaluation In Vitro/InVivo

Materials and Methods

1. Production, Selection and Purification of Anti-Tau Specific VHH(Tau-A2)

1.1 Antigen Preparation and Induction of a Humoral Immune Response inAlpaca

Subjects

Human cortical brain tissues from AD patients (Braak stage V and VI)were obtained from the NeuroCEB brain bank. This bank is associated to abrain donation program run by a consortium of patients associations(including France Alzheimer Association) and declared to the FrenchMinistry of Research and Universities, as requested by French Law. Anexplicit written consent was obtained for the brain donation inaccordance with the French Bioethical Laws.

Tissue extraction was performed according to Mercken et al. (1992 ActaNeuropathol., 84, 265-272). Cortex from AD brain (0.2 g) was homogenizedin 10 volumes of 10 mM Tris, 1 mM EGTA, 0.8 M NaCl pH7.4 containing 10%sucrose and was centrifuged at 27,000×g for 20 min at 4° C. The pelletwas removed and the supernatant was adjusted to 1% N-laurylsarcosine and1% beta-mercaptoethanol and incubated while rotating for 2.5 hours at37° C. The supernatant mixture was centrifuged at 100,000 g for 35 minat 20° C. The PHF containing pellet was gently washed with PBS andfinally resuspend in the same buffer.

One alpaca (Inti) was immunized with tau pellet and an other alpaca(Rascar) was immunized with the single phospho-peptide derived from theC-terminus of a tau protein of sequence CSIDMVDS(PO₃H₂)PQLATLAD (SEQ IDNO. 6), coupled to KLH (Eurogentec).

250 μl (500 μg) of both antigens was mixed with 250 μl of Freundcomplete adjuvant for the first immunization, and with 250 μl of Freundincomplete adjuvant for the following immunizations. After threeimmunizations at day 0, 21 and 40, a serum sample was taken at day 52and the immune response monitored by ELISA using recombinant phospho-tauprotein or recombinant non-phosphorylated tau protein.

1.2 Library Construction and Panning

250 ml of blood of the immunized animals was collected at day 52 and theperipheral blood lymphocytes isolated by centrifugation on a Ficoll(Pharmacia) discontinuous gradient and stored at −80° C. until furtheruse. Total RNA and cDNA was obtained as previously described in LafayeP. et al. (1995 Res Immunol., 146, 373-382), and DNA fragments encodingVHH domains amplified by PCR using CH2FORTA4 and VHBACKA6 primers, whichanneal to the 3′ and 5′ flanking region of the VH genes, respectively.The amplified product was used as template in a second round of PCRusing either of the primers VHBACKA4 and VHFOR36. The primers werecomplementary to the 5′ and 3′ ends of the amplified product andincorporated SfiI and NotI restriction sites at the ends of the VHHgenes. The PCR products were digested and ligated into phage expressionvector pHEN1. The resulting library was composed of two sub-libraries,one derived from tau pellet and the other from phospho peptide coupledto KLH. Phages were produced and isolated using both sub-libraries, andsubsequently pooled.

The library (>6×10⁸ clones) was panned against full-length phospho-tauprotein. Nunc Immunotubes (Maxisorp) tubes were coated overnight at 4°C. with the antigen (10μg/ml) in PBS. Phages (10¹² transducing unit)were panned by incubation with the coated tubes for 1 h at 37° C. withgentle agitation. A different blocking agent was used at each of thethree rounds of panning: 2% skimmed milk, Licor blocking buffer(Biosciences) diluted 1:4, and 4% BSA were respectively used. Phageclones were screened by standard ELISA procedures using a HRP/anti-M13monoclonal antibody conjugate (GE Healthcare) for detection (see below).The screening was performed in parallel with phospho-tau protein and tauprotein.

1.3 Expression of VHHs

The coding sequence of the selected nanobodies in vector pHEN1 wassub-cloned into a bacterial expression vector pET23d containing a6-Histidine tag using NcoI and NotI restriction sites. Transformed E.coli BL21 (DE3) pLysS cells express VHH in the cytoplasm after overnightinduction with IPTG (0.5 mM) at 16° C. Purified VHHs were isolated byIMAC from cytoplasmic extracts using a HiTrap crude column charged withNi²⁺ (GE Healthcare), according to the manufacturer's instructions. TheVHHs were eluted in 50 mM sodium phosphate buffer, 300 mM NaCl and 500mM imidazole buffer and dialyzed in PBS buffer containing 300 mM NaCl(PBS/NaCl).

The coding sequence of selected VHHs was also sub-cloned into a modifiedpASK IBA2 expression vector containing a Strep-tag at C-ter end (Skerraand Schmidt 1999 Biomol Eng., 16, 79-86). Same restriction sites wereused. E. coli XL2-Blue Ultracompetent cells were transformed to expressVHHs in the periplasm after overnight induction with anhydrotetracycline(AHT, 200 μg/l) at 16° C. VHHs were then purified from bacterial extractwith Strep-Tactin column (IBA) according to manufacturer's instructions.

2. VHH Tau-A2 Coupling to MRI Contrast Agents and to Fluorophores

2.1 General Synthesis Methods

Unless otherwise specified, the amino-acid derivatives and the reagentswere purchased from Novabiochem and Sigma-Aldrich, respectively. Theconcentration of the peptide and VHH solutions (net protein content) wasdetermined by quantitative amino acid analysis (AAA) using a Beckman6300 analyzer after hydrolysis of the compounds with 6N HCl at 110° C.for 20 hours. The RP-HPLC/MS analyses were performed on an Alliance 2695system coupled to a UV detector 2487 (220 nm) and to a Q-Tofmicro™spectrometer (Micromass) with an electrospray ionisation (positive mode)source (Waters). The samples were cooled to 4° C. on the autosampler.The linear gradient was performed with acetonitrile+0.025% formic acid(A)/water+0.04% TFA+0.05% formic acid (B) over 10 or 20 min. The columnused was a XBridge™ BEH300 C18 (3.5 μm, 2.1×100 mm) (Waters) (gradient10-100% A). The source temperature was maintained at 120° C. and thedesolvation temperature at 400° C. The cone voltage was 40 V. Thesamples were injected at 0.4-1 mg/ml concentration in their respectivebuffer added with B.

A maleimido-(DOTA/Gd)₃ compound 1 was prepared by solid-phase peptidesynthesis using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry as shown inFIG. 6.

2.2 Production of Tau-A2-SH

The coding sequence of a Cys-engineered VHH (Tau-A2-SH) was cloned intoa bacterial expression vector pET23d using NcoI and XhoI restrictionsites. To summarize, Tau-A2-SH (SEQ ID NO. 14) comprises from the N tothe C terminus a 6-Histidine tag, a thrombin cleavage site, VHH Tau-A2sequence followed by a G₃S spacer and three extra amino acids CSA.Transformed E. coli BL21 (DE3) pLysS cells express Tau-A2-SH in thecytoplasm after overnight induction with IPTG (0.5 mM) at 16° C.Purified VHHs were isolated by IMAC from cytoplasmic extracts using aHiTrap crude column charged with Ni²⁺ (GE Healthcare), according tomanufacturer's instructions. The protein was eluted in PBS/NaClcontaining 500 mM imidazole and then dialyzed in PBS/NaCl.

AAA: Ala 14.3 (14), Arg 9.6 (10), Asp+Asn 7.3 (6), Glu+Gln 11.2 (10),Gly 18.4 (19), His 5.7 (6), Ile 4.2 (4), Leu 8.6 (8), Lys 5.3 (5), Phe 4(4), Pro 2.9 (2), Ser 18.9 (23), Thr 10.5 (12), Tyr 6.1 (7), Val 11.5(11).

MS: 15499.286 (C669H1047N205O213S4 calculated 15498.190). The MScorresponds to the protein with N-ter deleted methionine.

2.3 Synthesis of Tau-A2-S-Alexa Fluor® 488 (Tau-A2-S-AF488)

The cysteine present in the C-ter tripeptide of Tau-A2-SH was used tocouple the Tau-A2-SH to the maleimido Alexa Fluor® 488 fluorophore(Invitrogen). pH of the solution containing Tau-A2-SH was adjustedbetween 6.8 and 7. The solution was then gently stirred with 10-foldmolar excess of tris(2-carboxyethyl) phosphine (TCEP) at roomtemperature for 30 min, to allow complete reduction of anyintermolecular disulfide bond. A 10-fold molar excess of maleimido AlexaFluor® 488 fluorophore dissolved in dimethylformamide (DMF) was added.Notably, for high efficiency of conjugation, the volume percentage ofDMF in the final solution was kept below 5%. The conjugation wasperformed for 2 hours at room temperature, under protection from light.The non-conjugated maleimido fluorophore was then removed by successivedialysis with PBS containing 300 mM NaCl (overall concentration).

2.4 Synthesis of Tau-A2-S-(DOTA/Gd)₃

Prior to the conjugation, Tau-A2-SH (4.2 ml, 0.32 mg/ml in PBS/NaCl pH6.8) was treated with TCEP (24.6 μg, 5 eq) for 30 min to prevent thedimerization of the VHH. Maleimido-(DOTA/Gd)₃ (0.78 mg, 3.8 eq relativeto 1 thiol group per VHH) in aqueous solution (78 μl) was added to theprotein and the solution was stirred at 4° C. for 3 h. The solution wasthen dialyzed in PBS/NaCl using Slide-A-Lyzer cassettes (ThermoScientific) (3,500 MWCO). Aliquots (20 μl) of Tau-A2-SH andTau-A2-S-(DOTA/Gd)₃ were diluted with buffer B (20 μl) for RP-HPLC/MSanalyses. Further, aliquots (10 μl) of the same compounds were dilutedin 20 mM Tris buffer pH 7.3 (90 μl) for ELISA analyses. 4.2 ml ofTau-A2-S-(DOTA/Gd)₃ (0.24 mg/ml) was obtained with a yield of 65%. Itwas calculated by dividing the actual amount of the final productTau-A2-S-(DOTA/Gd)₃ by its expected amount (net protein contents). Forfurther experiments, the solution of Tau-A2-S-(DOTA/Gd)₃ wasconcentrated four times using Vivaspin 2 centrifugal filter device(3,000 MWCO PES).

AAA: Ala 13.4 (14), Arg 9.6 (10), Asp+Asn 8.0 (6), Glu+Gln 11.8 (10),Gly 24.4 (22), His* (6), Ile 4.2 (4), Leu 8.6 (8), Lys 18.9* (8), Phe 4(4), Pro 2.9 (2), Ser 16.8 (23), Thr 10.7 (12), Tyr 6.1 (7), Val 11.4(11). [*His cannot be determined due to co-elution with ammonium. Lys isoverestimated due to co-elution with maleimido derivative in theconditions of the analysis.].

MS: 17887.549 (C751H1174N227O244S4Gd3 calculated 17886.980).

3. In Vitro Characterization of Tau-A2, and Tau-A2 Conjugates byImmunohistochemistry and Biochemistry

3.1 Subjects

Human brain tissue was obtained from the NeuroCEB brain bank.Preclinical experiments were performed on Tg4510 (Santacruz et al. 2005Science, 309, 476-81) transgenic mice. Animal experimental procedureswere performed in strict accordance with the ethical standards of Frenchand European laws (European Communities Council Directive 2010/63/EU onthe protection of animals used for scientific purposes) and afterapproval from local Animal Care and Use committee. The animals weresacrificed using a high dose of sodium pentobarbital (100 mg/kg) andthen perfusion-fixed with 10% buffered formalin. Their brains were thenremoved, immersed in formalin for at least 24 hours and stored at 4° C.

3.2 Tissue Extracts

Tissue extraction was performed according to Mercken et al. (1992 ActaNeuropathol., 84, 265-272).

3.3 Immunoblots

Phospho-tau protein was resuspended in NuPAGE® LDS sample buffer(Invitrogen). Brain extracts were resuspended in NuPAGE® LDS samplebuffer (Invitrogen) containing 8 M urea. Following separation bypolyacrylamide gel electrophoresis (PAGE) using NuPAGE Novex 4-12%Bis-tris gel (Invitrogen), semi-dry transfer onto Hybond-C (Amersham)and western blotting were carried out using the Xcell II blot module(Invitrogen). Prior to the immunochemical reaction, membranes wereblocked in a 4% skimmed milk solution. Immunoblotting of membranes wasaccomplished with VHH (with His or Strep tag) or anti p-tau 422 mAb(Grueninger et al. 2011 Mol Cell Biochem., 357, 199-207) and revealed byrabbit anti-His tag (eBioscience) polyclonal antibodies followed byperoxidase labeled goat anti-rabbit immunoglobulins (Abcam) or by ananti-Strep tag monoclonal antibody (such as the antibody C23-21 producedby the hybridoma filed with the CNCM under the number I-4703) followedby peroxidase labeled rabbit anti-mouse immunoglobulins (Bio-rad).Finally, peroxidase activity was visualized using a chemiluminescent kit(GE Healthcare).

3.4 ELISA

Microtiter plates (Nunc, Denmark) were coated by incubation overnight at4° C. with 1 μg/ml of phospho-tau or tau protein or the phospho-peptideof SEQ ID NO. 6 coupled to ovalbumine protein diluted in PBS. Plateswere washed with buffer 0.1% Tween 20 in PBS. Tau-A2 (with His or Streptag) was diluted in buffer 0.5% gelatin 0.1% Tween 20 in PBS. After 2 hincubation at 37° C., plates were washed again before addingrespectively a rabbit anti-His tag polyclonal antibody (eBiosciences),followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abcam)or by an anti-Strep tag monoclonal antibody (such as the antibodyC23-21) followed by peroxidase labeled rabbit anti-mouse immunoglobulins(Bio-rad), and finally revealed by OPD (o-phenylendiaminedihydrochloride, Dako) according to manufacturer's protocol.

3.5 Sequences Analysis

VHH encoded DNAs were sequenced by GATC Biotech and sequences weretreated with Serial Cloner.

3.6 Determination of pI

The pI of VHHs was determined by isoelectric focusing using IEF 2-9 Gel(Invitrogen). NEPGHE (non equilibrium pH gradient gel electrophoresis)with sample application at the anode because it allows optimal proteinanalysis in the basic range of the gel including pH 8.5 to 10.5. Theprotocol was detailed in SERVAGel IEF 3-10 instruction manual.

3.7 Immunohistochemistry and Immunofluorescence

Immunohistochemistry was performed on formalin-fixed tissues(paraffin-embedded or frozen sections or vibratome sections). StandardIHC protocols were applied and adapted for each tissue conditions. Asmost of immunostaining experiments were performed using paraffinsections, a detailed protocol for paraffin-embedded material isdescribed herein. Immunostaining of brain tissue was performed on 4 μmthick paraffin sections. Both human and mouse tissues were used (Humanpatients with AD or other tauopathies and Tg4510 mice (Santacruz et al.2005 Science, 309, 476-481). Sections were de-paraffinized in xylene,rehydrated through ethanol (100%, 90%, and 70%), 5 min for each solutionand finally brought to running tap water for 10 min. They were thenincubated in 98% formic acid for 5 min, washed again under running tapwater, quenched for endogenous peroxidase with 3% hydrogen peroxide and20% methanol, and finally washed in water. Non-specific binding wasblocked by incubating the sections for 30 min in 2% bovine serum albuminin TBS+0.5% Tween. Appropriate dilutions of primary antibodies (1-10μg/ml of VHH with either His or Strep tag) were then applied and slicesincubated overnight in a humidified chamber at 4° C. Slides were washedwith TBS-Tween and incubated with secondary antibodies rabbit anti-HisTag for 1/1000 or home-made biotinylated anti-strep mAb C23-21 inTBS-Tween at room temperature for 1 hour. Slides were then incubatedwith reagents of Dako REAL™ Detection System, Peroxidase/DAB+ accordingto manufacturer's instructions. Chromogenic (DAB) revelation wasdeveloped until a good signal-to-noise ratio was obtained (about 5 min).After washing with TBS-Tween, slides were counter-stained withhematoxylin. For labeling of NFTs, biotinylated mAb AT8(ThermoScientific) was used as a positive control in parallel.

Immunofluorescent staining of NFTs using Tau-A2-S-AF488 was performed on40 m thick free floating sections obtained from Tg4510 mice usingvibratome (Leica VT1000S). After 3×5 min washing in PBS, sections wereblocked by incubating for 15 min with PBS-triton 0.2% containing 2% ofBSA, which was then replaced by 1 μg/ml of Tau-A2-S-AF488 in PBS-triton0.2% and incubated overnight at 4° C. Sections were finally washed withPBS and mounted with an aqueous mounting medium (Mowiol).

4. In Vivo Evaluation of Tau-A2 and Tau-A2 Conjugates by Two-PhotonImaging and Correlative Immunohistochemistry

4.1 Subjects

In vivo evaluation of Tau-A2, and Tau-A2-S-(DOTA/Gd)₃ was performed onTg4510 transgenic mice.

4.2 Stereotaxic Injection of VHH and IHC

Stereotaxic injections were performed in Tg4510 female transgenic mice(n=2) anesthetized mice with 2 μl of VHH per injection at the rate of0.5 μl/min. The mice were anesthetized with a mixture of isoflurane(1-2%) and air (1 l/min). They were placed on a stereotaxic frame andthe skull was bilaterally perforated with a Dremel. Blunt Hamiltonsyringes were used to inject MRI contrast agent. Each mouse received 4injections, in the frontal cortex and the hippocampus in eachhemisphere. The stereotaxic coordinates in the frontal cortex were +0.86mm anterior from bregma, ±1.5 mm lateral from the midline, −0.65 mmventral from dura. The stereotaxic coordonates in the hippocampus were−2.18 mm posterior from bregma, ±1.5 mm lateral from the midline, −1.8mm ventral from dura. Two or 24 hours after the injection, mice wereeuthanized and perfused intracardially with 4% paraformaldehyde in PBS(pH 7.6). Brains were removed and post-fixed in the same fixativeovernight at 4° C. 4 μm thick paraffin sections were prepared. Thepresence of the VHH in cerebral tissue was detected usingimmunohistochemical procedures described above.

4.3. Two Photon Microscopy in Tg4510 Mice Using Tau-A2-S-AF488

1) Craniotomy

Two 8-month-old Tg4510 mice were anesthetized by inhalation ofisoflurane (1% vol/vol in pure O₂) and placed onto a warming blanket(37° C.). A stereotaxic frame was used to identify the location of themotor cortex. 50 μl of 2% lidocaine was injected subcutaneously forlocal anesthesia at the incision site location where the skin was to beremoved. A less than 2 mm craniotomy was performed using a scalpel. Inthe case of intracerebral injections of VHH Tau-A2-S-AF488, the duramater was incised. In the case of intravenous injections, the dura materwas kept intact. To avoid movement artifacts, the skull opening wascovered with 2% low melting point agarose and a coverglass. The opticalwindow was secured and sealed to skull with dental cement, covering allthe exposed skull, wound margins and coverglass edges.

2) Administration of Tau-A2-S-AF488

Intracerebral injection: 1.2 μg (1.5 μL) of Tau-A2-S-AF488 was injectedin the brain of one Tg4510 mouse at 1.5 mm depth from cortical surface.Two-photon imaging was then performed during 4 h after injection.

Intravenous injection: 270 μg (150 μl) of Tau-A2-S-AF488 was slowlyinjected into the caudal vein of one Tg4510 mouse. Two-photon imagingwas then performed in the following 3 hours.

3) Two-Photon Imaging

Two-photon imaging was performed with a two-photon laser-scanningmicroscope system and PrairieView software (Prairie Technologies,Middelton, Wis., USA), using a 16×0.9 NA water immersion objective(Nikon, Tokyo, Japan) with the 2-photon laser tuned to 920 nm (MaiTaiDeepSee, Spectra Physics, Mountain View, Calif., USA). The images wereacquired at 512×512 with a pixel size of 0.5 μm. Care was taken to useless than 20 mW of laser power in the tissue.

5. Obtention of Rabbit Polyclonal Anti-VHH Antibodies

Purified alpacas immunoglobulins were used to immunize one rabbit.Rabbit polyclonal antibodies against VHH were purified in 2 steps. Firstpolyclonal antibodies were isolated by immunochromatography usingprotein A-sepharose 4B beads. Then another immunochromatogaphy wasrealized by using sepharose 4B beads labelled with Tau-A2var-SH. Thesepharose labelling was realized according to manufacturer'sinstructions (GE). The resulting purified antibodies represented lessthan 1% of total rabbit antibodies and are referred to as rabbitpolyclonal anti-VHH antibodies. These antibodies bind to Tau-A2 VHH.

Results

1. Polyclonal Response, Library Construction, and Selection of SpecificAnti-Tau VHH

After 3 immunizations, the sera of Inti (brain extract) and Rascar(tau-pS422) were collected and their binding to phospho-tau and nonphosphorylated tau was analyzed by ELISA (FIG. 1). The sera of Inti andRascar recognized both phosphorylated tau and non phosphorylated tau,although the immune response of Inti appeared to be better than that ofRascar. However, the former did not distinguish significantlyphospho-tau and non phospho-tau and the latter showed a higher immuneresponse for phospho-tau.

In order to ensure that one or some of anti phospho-tau antibodiespresent in the sera could recognize tau lesions in fixed mouse tissues,IHC was performed on both frozen microtome floating sections andnon-frozen vibratome floating sections from TauPS2APP mice (GrueningerF. et al., 2010, Neurobiol Dis., 37:294-306). The serum of Inti did notshow any immune staining of tau lesions but otherwise the serum ofRascar demonstrated a specific tau staining of NFTs on both microtomeand vibratome sections (data not shown).

Despite the absence of immunodetection for NFTs in IHC with Intipolyclonal serum, it can not be excluded the existence of phospho-tauspecific antibodies in its blood plasma. Indeed, the lack of IHC signalcould be due to the low frequency of anti phospho-tau antibodies, whichcould recognize the epitopes of phospho-tau present in NFTs of TauPS2APPmice. The two libraries obtained from these two animals were pooled forpanning experiments.

Total RNA from peripheral blood lymphocytes was used as template forcDNA synthesis. Using this cDNA, the VHH encoding sequences were thenamplified by PCR and cloned into vector pHEN1. Subsequenttransformations yielded two libraries of about 3×10⁸ clones each. Bothlibraries were pooled and VHHs displaying the best affinity wereselected by phage display through 3 panning cycles with phosphorylatedtau. 96 individual clones were tested by ELISA on phosphorylated tau andon tau proteins. Only one clone was found to bind specifically onphosphorylated tau (VHH Tau-A2).

This VHH was subcloned in vector pET23 or in vector pASK IBA2 to allow ahigh level of expression of VHH with, respectively, a His-tag or aStreptavidin-tag. Yields of <1 mg/l of bacterial culture were obtained.The single domain products were shown to be pure to homogeneity bySDS-PAGE and by RP-HPLC/MS (data not shown); its pI values was above9.5. Dynamic light scattering experiments showed that Tau-A2 ismonomeric (RH=4.5±0.3 nm) and is not aggregated after purification.Amino-acid sequence of VHH Tau-A2 is referred to as SEQ ID NO. 4.

2. Recognition of NFTs by VHH Tau-A2

2.1 VHH Tau-A2 Recognizes the Phosphorylated Serine 422 in the C-Ter TauPeptide

Several serine and threonine are phosphorylated on the pathogenic formof tau. To evaluate the role of phosphorylation in the epitoperecognition, ELISA was performed on phosphorylated tau and singlephospho-peptide derived from the C-terminus of a tau protein (sequenceCSIDMVDS(PO₃H₂)PQLATLAD; SEQ ID NO. 6), coupled to ovalbumin (Ova)protein. Phosphorylated tau was the full-length tau proteinphosphorylated at multiple sites including S422. VHH Tau-A2 binds bothcompounds suggesting that VHH Tau-A2 recognized an epitope including thephospho serine 422 (pS422) (FIG. 2).

2.2 Immunoreactivity of VHH Tau-A2 for NFTs

The distribution of VHH-specific immunoreactivity was examined in humanAD brains, in tissues from other tauopathies (fronto-temporal dementia[FTD], progressive supranuclear palsy [PSP] and Pick's disease [PD]) andin transgenic Tg4510 mice brains.

VHH Tau-A2 showed good ability to immunodetect NFTs in paraffin sectionsfrom AD patients (FIG. 3A). Also tau-positive glial inclusions wereobserved in the FTD case (oligodendroglial coiled bodies) and in the PSPcase (astrocytic tufts) (FIG. 3B). Interestingly the typical cellularinclusions (Pick bodies) identified by the reference anti-tau AT8 mAbwere negative for Tau-A2 (FIG. 3C) underlining some specificity of theVHH. As opposed to other tauopathies Pick bodies are only constituted of3R tau proteins. Hence it can be hypothesized that Tau-A2 mainlyrecognizes tau with 4 repeats. No labeling was observed with tissuesfrom wild type mice.

Paralleling result on paraffin-embedded tissues, it was showed that NFTimmunodetection using VHH Tau-A2 can be readily obtained on mousefree-floating vibratome sections (FIG. 4).

To confirm the immunoreactivity of VHH Tau-A2 on brain tissues,western-blot immunoassays were performed on brain extracts obtained fromAD patients. Tau-pS422 mAb (Grueninger et al. 2011 Mol Cell Biochem.,357, 199-207) was used as a reference antibody. Recombinant phospho-tau(p-tau) was loaded in parallel on SDS-PAGE gel. Phospho-tau protein wasrevealed by both VHH Tau-A2 and Tau-pS422 mAb. One main band,corresponding to phosphorylated tau between 70 and 100 kDa, wasimmunodetected with Tau-A2 and Tau-pS422 in AD extracts. With Tau-pS422mAb, two additional bands with a molecular weight between 130 and 150kDa were observed indicated the presence of aggregated tau-pS422 in ADbrain, which was very slightly revealed by Tau-A2 (FIG. 5).

3. Cys-Engineered Tau-A2 and Antibody Coupling to Alexa Fluor® 488Maleimide

Cys-engineered Tau-A2 containing from the N to the C terminus a6-Histidine tag, a thrombin cleavage site, VHH Tau-A2 sequence followedby a G3S spacer and three extra amino acids CSA was cloned in vectorpET23d to allow a high level of expression (referred to as Tau-A2-SH orA2-SH; SEQ ID NO. 14). Tau-A2-SH was conjugated to maleimido AlexaFluor® 488 by thioaddition. The resulting product Tau-A2-S-AF488 wasanalyzed by SDS-PAGE, IEF/NEPGHE (FIGS. 7A and 7B) and RP-HPLC/MS. Itwas shown to be pure to homogeneity by SDS-PAGE and by RP-HPLC/MS. Allanalyses showed that Tau-A2-SH was totally converted into thewell-defined conjugate, referred to as Tau-A2-S-AF488, with a singleAF488 on the VHH Tau-A2. Tau-A2-SH possesses a pI around 10 (between 9.5and 10.5) and this value slightly decreases after AF488 conjugation,still remaining >9.5 (see FIG. 7B). The hydrodynamic radius (R_(H)) ofTau-A2-SH and Tau-A2-S-AF488 was separately measured by DLS. The sizedistribution over time showed an average R_(H) of 2.66±0.0788 nm forTau-A2-SH and an average R_(H) of 3.576±0.225 nm for Tau-A2-S-AF488,which suggested that both of them were in monomeric form in solution.

4. Detection of NFTs with VHH Tau-A2-S-AF488

4.1 In Vitro Labeling on Brain Slices of Tg4510 Mouse

After direct incubation with mouse brain sections, Tau-A2-S-AF488 showedgood ability to detect NFTs in Tg4510 mice by standard IHC (FIG. 7C-D),and direct fluorescent staining (FIG. 7E-F).

4.2 Two-Photon Imaging after Intracerebral Injection

In vivo two-photon imaging of Tau-A2-S-AF488 was performed after directintracerebral injection of 1.2 μg (1.5 μL) in a Tg4510 mouse brainfollowing a craniotomy and perforation of the dura mater. A large amountof CSF was observed and a high auto-fluorescence (even before theinjection) was observed. Despite these limitations for appropriatebi-photon imaging, specific staining of NFTs was detected after topiccortical injection (FIG. 8).

4.3 Two-Photon Imaging after Intravenous Injection of VHH Tau-A2-S-AF488

A 10 mg/kg dose of Tau-A2-S-AF488 was injected in the tail vein (270 g,150 μL) of two Tg4510 mice. BBB integrity of these mice was previouslychecked by MRI and absence of signal modification in the mice suggestedno disruption of BBB (data not shown). The conjugate extravasation andstaining in the brain was recorded for 4 hours post injection usingtwo-photon microscopy on brain window (z=from the surface up to 360 μmdeep). FIG. 9A displayed in vivo imaging (Maximum IntensityProjection—MIP) of Tau-A2-S-AF488 over time up to 180 min in the sameregion. Few seconds after iv injection, strong staining of arborescentvessels was observed and declined dramatically 20 min later with onlyfew capillary vessels remaining stained. This suggested a shorthalf-life of conjugated VHH in the circulation (10-20 min). 120 minafter iv injection, NFTs began to be visualized. The absence of signalin the red channel demonstrated that the fluorescent signal was specific(data not shown) and not due to general autofluorescence. VHHTau-A2-S-AF488 extravasation and staining in the brain was registeredfor 3 hours post injection using two-photon microscopy on brain window(from the immediate cortical surface up to 350 μm deep). In addition tostrong auto-fluorescence background and large amount of CSF (see above),the Tg4510 mice exhibited marked cerebral atrophy. A few seconds afteriv injection, strong staining of densely-packed vessels was observed.Specific tau staining of NFTs was observed 2 h after injection despiteremaining autofluorescence signal. A persistent and specific labeling ofNFTs was observed even 3 hours after injection (FIG. 9A), suggesting abrain half-life of Tau-A2-S-AF488 extending over several hours.

Four hours after the intravenous injection of Tau-A2-S-AF488, the brainwas harvested and 5 m-thick paraffin sections were prepared. IHC wasthen performed with rabbit polyclonal anti-VHH antibodies to confirm thediffusion and labeling of NFTs by Tau-A2-S-AF488 (FIG. 9B).

6. Control Experiments

Evaluation in NFTs-Free Mouse

Tau-A2-S-AF488 was intravenously injected in a wild type, NFT-free,C57BL/6 mouse.

No specific in vivo staining in the brain parenchyma was observed usingtwo-photon microscopy assay (data not shown).

Comparison with Conventional IgG Antibody

Injection of AF488-conjugated anti-Tau mAb (Grueninger et al. 2010Neurobiol. Dis., 37, 294-306) iv in a Tg4510 mouse did not allowdetection of NFTs indicating no significant extravasation of thisstandard anti-Tau-pS422 immunoglobulin.

7. Conclusion

Using two-photon microscopy after iv injection, VHH Tau-A2-S-AF488showed its ability to cross the BBB and to penetrate into neurons aftercrossing a second (plasma membrane) barrier to reach its cytoplasmictarget. Long-term detection (3 h) of NFTs labeling suggested also a longhalf-life of this VHH in the brain.

8. Antibody Coupling to MRI Contrast Agent

VHH Tau-A2-SH was labeled with gadolinium (MRI contrast agent) using1,4,7,10 tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as thechelating agent. A site specific coupling strategy was used whichinvolves the thioaddition of Tau-A2-SH to the syntheticmaleimido-(DOTA/Gd)3 compound. Tau-A2-SH was totally converted into thewell-defined conjugate Tau-A2-S-(DOTA/Gd)₃, as shown by RP-HPLC/MS, with65% yield. The pI of Tau-A2-S-(DOTA/Gd)₃ was slightly reduced comparedto the one of the unlabeled Tau-A2-SH (data not shown).

EXAMPLE II Synthesis of a Variant of Tau-A2-SH: Tau-A2VAR-SH

VHH Tau-A2 specifically detected phosphorylated Tau but its productionlevel is rather low, in average 150 μg/L, and it tends to aggregate. Itwas therefore needed to provide a variant of VHH Tau-A2 with improvedproperties.

Two hydrophobic amino acids located in 2 hydrophobic areas of VHHTau-A2-SH of SEQ ID NO. 14 were mutated: isoleucine at position 76replaced by a glycine (176G) and valine at position 110 by a glycine(V110G) (FIG. 10). These mutations increased the hydrophilicity of theVHH with an increase of the GRAVY index from −0.280 for Tau-A2-SH to−0.365 for the variant (GRAVY or Grand average of hydropathicity indexindicates the solubility of the proteins: positive GRAVY (hydrophobic),negative GRAVY (hydrophilic)). Glutamine at position 26 was also mutatedby a glutamic acid (Q26E) and an aspartic acid (D) and anvaline (V) wereadded between position 22 and 23 to slightly decrease the pI (lessbasic) (FIG. 10). Three of these mutated amino acids (D23, E26, andG110) are located outside the CDRs. This mutant is referred to asTau-A2var-SH (SEQ ID NO. 17).

VHH Tau-A2var-SH (SEQ ID NO. 17) was cloned in pET23d vector andexpressed in E. coli. It was purified on a Ni-NTA resin by elution inimidazole. Analyses of Tau-A2-SH in comparison with Tau-A2var-SH wereperformed by several methods:

-   -   SDS-PAGE showed only one band at an apparent molecular weight        around 15-16 KDa (FIG. 11A). A better production of Tau-A2var-SH        was observed with an expression rate estimated to 0.8-1 mg/L, 5        times more than the parental counterpart. Moreover Tau-A2var-SH        has a lower tendency to aggregate. The pI is 9.68 slightly less        basic than Tau-A2-SH (FIG. 11B).    -   the ability of both VHHs to recognize phospho-Tau was evaluated        by ELISA (FIG. 11C). Comparison of the binding curves of        Tau-A2var-SH and Tau-A2-SH did not show any difference for their        recognition of phospho-Tau (FIG. 11C).    -   to further assess the functional activity of Tau-A2var-SH, IHC        was then carried out on tissue from mice model of NFTs (Tg4510        mice). After incubation of the brain slices with the different        compounds and revelation by an anti-His secondary antibody, a        detection of NFTs was observed with both compounds on mouse        tissues (FIG. 11D). Immunostaining was even slightly enhanced        using Tau-A2var as compared to Tau-A2.

The VHH Tau-A2var-SH was labeled using a similar procedure on thenon-dialized protein solution, directly after the affinity columnelution.

Then the ability of in vivo detection of NFTs with VHHTau-A2var-S-(DOTA/Gd)₃ have been realized. A 10 mg/kg dose ofTau-A2var-S-(DOTA/Gd)₃ was injected in the tail vein (270 μg, 150 μL) oftwo Tg4510 mice. Four hours after the intravenous injection ofTau-A2var-S-(DOTA/Gd)₃, the brain was harvested and 5 m-thick paraffinsections were prepared. IHC was then performed with rabbit polyclonalanti-VHH antibodies. NFTs were labeled in the cortex (FIG. 12). Thesedata confirm the brain penetration, diffusion and labeling of NFTs byTau-A2var-S-(DOTA/Gd)₃.

CONCLUSION

These results show that Tau-A2var behaves in a similar way as Tau-A2 forthe recognition of phospho tau and NFTs. Using IHC studies after ivinjection, VHH Tau-A2var-S-(DOTA/Gd)₃ showed its ability to cross theBBB and to penetrate into neurons after crossing a second (plasmamembrane) barrier to reach its cytoplasmic target.

1. An isolated variable domain of a camelid heavy-chain antibody (VHH),characterized in that it is directed against a phosphorylated tauprotein and it is obtainable by immunizing a camelid with the singlephospho-peptide derived from the C-terminus of a tau protein of sequenceCSIDMVDS(PO₃H₂)PQLATLAD (SEQ ID NO. 6) or a phospho-tau enrichedAlzheimer's disease brain extract from a human.
 2. The VHH according toclaim 1, characterized in that the VHH binds the phosphorylated serine422 of a phosphorylated tau protein.
 3. The VHH according to claim 1 orclaim 2, characterized in that the method comprises the steps of: (a)immunizing a camelid, preferably a Lama pacos, with the singlephospho-peptide derived from the C-terminus of a tau protein of sequenceCSIDMVDS(PO₃H₂)PQLATLAD (SEQ ID NO. 6) or a phospho-tau enrichedAlzheimer's disease brain extract from a human, (b) isolating peripherallymphocytes of the immunized camelid, obtaining the total RNA andsynthesizing the corresponding cDNAs, (c) constructing a library of cDNAfragments encoding VHHs, (d) transcribing the VHH-encoding cDNAsobtained in step (c) to mRNA using PCR, converting the mRNA to ribosomedisplay format, and selecting the VHH by ribosome display, and (e)expressing the VHH domain in a vector.
 4. The VHH according to any oneof claims 1 to 3, characterized in that the camelid is a Lama pacos. 5.The VHH according to any one of claims 1 to 4, characterized in that itsamino acid sequence comprises, from the N-terminus to the C-terminus,the amino acid sequence SEQ ID NO. 1, the amino acid sequence SEQ ID NO.2 and the amino acid sequence SEQ ID NO.
 3. 6. The VHH according toclaim 5, characterized in that it consists of the amino acid sequenceSEQ ID NO. 4 or SEQ ID NO.
 5. 7. An isolated variant of the VHH of SEQID NO. 5, wherein said VHH variant is directed against thephosphorylated serine 422 of a phosphorylated tau protein, and whereinthe amino acid sequence of said variant has at least 95% identity withthe amino acid sequence SEQ ID NO:
 5. 8. The isolated variant accordingto claim 7, characterized in that its amino acid sequence comprises,from the N-terminus to the C-terminus, the amino acid sequence SEQ IDNO. 1, the amino acid sequence SEQ ID NO. 2 and the amino acid sequenceSEQ ID NO.
 3. 9. The isolated variant according to claim 7,characterized in that has the amino acid sequence SEQ ID NO. 5 havingthe following mutations: the Glutamine residue (Gln, Q) at position 3 ofthe amino acid sequence SEQ ID NO. 5 is substituted with an amino acidresidue selected from the group consisting of Aspartic acid (Asp, D) andGlutamic acid (Glu, E), preferably Glu, the Isoleucine residue (Ile, I)at position 52 of the amino acid sequence SEQ ID NO. 5 is substitutedwith an amino acid residue selected from the group consisting of Alanine(Ala, A), Glycine (Gly, G) and preferably Gly, the Valine residue (Val,V) at position 86 of the amino acid sequence SEQ ID NO. 5 is substitutedwith an amino acid residue selected from the group consisting of Alanine(Ala, A), Serine (Ser, S), Threonine (Thr, T), Asparagine (Asn, N),Glutamine (Gln, Q), Aspartic acid (Asp, D), Glutamic acid (Glu, E),Lysine (Lys, K), Arginine (Arg, R) and Glycine (Gly, G), preferably Gly,and optionally two amino acid residues are added in N-terminal positionof the amino acid sequence SEQ ID NO. 5 and are selected from the groupconsisting of the dipeptides Glutamic acid-Valine (E-V) and Asparticacid-Valine (D-V), preferably D-V.
 10. The isolated VHH variantaccording to claim 7, characterized in that it consists in the aminoacid sequence SEQ ID NO. 15 or SEQ ID NO.
 16. 11. A VHH derivativeconsisting of a polypeptide comprising a VHH according to any one ofclaims 1 to 6 or a VHH variant according to any one of claims 7 to 10,provided that said VHH or VHH variant comprised in said polypeptide isable to bind a phosphorylated tau protein.
 12. A VHH derivativeaccording to claim 11, characterized in that it has the formula P-C-Z orZ-C-P, wherein: P is a 100-500 amino acid peptide comprising orconsisting of a VHH according of any one of claims 1 to 6 or a VHHvariant according to any one of claims 7 to 10, wherein said amino acidsequence has no accessible reduced cystein residue, and preferably hasno reduced cystein residue, C is a cystein residue, Z represents a 1-10amino acid spacer, preferably a 1-10 neutral or negatively charged aminoacid spacer, wherein the amino acid residues of Z are identical ordifferent and wherein Z does not contain a cystein residue.
 13. Anisolated polynucleotide encoding a VHH of any one of claims 1 to 6 or aVHH variant of any one of claims 7 to 10 or a VHH derivative of any oneof claims 11 or
 12. 14. A recombinant expression cassette comprising apolynucleotide of claim 13 under the control of a transcriptionalpromoter allowing the regulation of the transcription of saidpolynucleotide in a host cell.
 15. A recombinant vector comprising apolynucleotide of claim 13 or a recombinant expression cassette of claim14.
 16. A host cell containing a recombinant expression cassette ofclaim 14 or a recombinant vector of claim
 15. 17. A diagnostic ortherapeutic agent comprising a VHH of any one of claims 1 to 6 or a VHHvariant of any one of claims 7 to 10 or VHH derivative of any one ofclaims 11 or 12, linked, directly or indirectly, covalently ornon-covalently to a substance of interest.
 18. The diagnostic ortherapeutic agent according to claim 17, characterized in that the VHHderivative is as defined in claim 12 and in that the cystein residue Cof said VHH derivative is linked to a substance of interest through asulfide bond.
 19. The diagnostic or therapeutic agent according to claim18, characterized in that said cystein residue C is linked to asubstance of interest through a thiol-reactive compound bearing saidsubstance of interest.
 20. The diagnostic or therapeutic agent accordingto claim 19, characterized in that the thiol-reactive compound is amaleimido compound.
 21. The therapeutic agent according to any one ofclaims 17 to 20, characterized in that the substance of interest isselected from a peptide, an enzyme, a nucleic acid, a virus and achemical compound.
 22. The therapeutic agent according to any one ofclaims 17 to 21, characterized in that the therapeutic compound isselected from the group consisting of an analgesic compound, ananti-inflammatory compound, an antidepressant compound, a cytotoxiccompound, an anticonvulsant compound or an anti-neurodegenerativecompound.
 23. The diagnostic agent according to any one of claims 17 to20, characterized in that the diagnostic compound is selected from thegroup consisting of an enzyme, a fluorophore, a NMR or MRI contrastagent, a radioisotope, or a nanoparticle.
 24. The diagnostic agent ofclaim 23, characterized in that the diagnostic compound is a NMR or MRIcontrast agent selected from the group consisting of paramagnetic agentsgadolinium (Gd), dysprosium (Dy) and manganese (Mn), and thesuperparamagnetic agents based on iron oxide or iron platinum, andX-nuclei such as ¹⁸F, ¹³C, ²³Na, ¹⁷O, ¹⁵N, preferably gadolinium. 25.The diagnostic agent of claim 23, characterized in that the diagnosticcompound is a fluorophore selected from the group consisting of a greenfluorescent protein (GFP), blue fluorescent dyes excited at wavelengthsin the ultraviolet (UV) part of the spectrum, AMCA(7-amino-4-methylcoumarin-3-acetic acid), Alexa Fluor® 350, greenfluorescent dyes excited by blue light, FITC, Cy2, Alexa Fluor® 488, redfluorescent dyes excited by green light, rhodamines, Texas Red, Cy3,Alexa Fluor® dyes 546, 564 and 594, dyes excited with far-red light andCy5, preferably Alexa Fluor®
 488. 26. The diagnostic or therapeuticagent of claim 17, characterized in that the substance of interest is aliposome or a polymeric entity comprising a diagnostic or therapeuticcompound as defined in any of claims 21 to
 25. 27. Non-site specificmethod for coupling a VHH of any one of claims 1 to 6 or a VHH variantof any one of claims 7 to 10 or a VHH derivative of any one of claims 11or 12 with a substance of interest, said method comprising a conjugationstep of a substance of interest with a VHH of any one of claims 1 to 6or a VHH variant of any one of claims 7 to 10 or a VHH derivative of anyone of claims 11 or
 12. 28. Site specific method for coupling asubstance of interest with a VHH derivative of claim 12 comprising aconjugation step of said VHH derivative with a substance of interestbearing a thiol-reactive function.
 29. A kit for brain imaging, or fordiagnosing or monitoring a disorder mediated by neurofibrillary tangles,neuropil threads or dystrophic neurites, such as tauopathies, includingAlzheimer's disease (AD), Pick disease (PD), fronto-temporal dementia(FTD), corticobasal degeneration (CBD) and progressive supranuclearpalsy (PSP), comprising either at least a VHH of any one of claims 1 to6 or a VHH variant of any one of claims 7 to 10 or a VHH derivative ofany one of claims 11 or 12 and a diagnostic agent and optionally adiagnostic reagent, or a diagnostic agent of any one of claims 17-18 and21-24 and a diagnostic reagent.
 30. Use of a diagnostic agent of any oneof claims 17-20 and 23-26 for diagnosing or monitoring a disordermediated by neurofibrillary tangles, neuropil threads or dystrophicneurites, such as tauopathies, including Alzheimer's disease (AD), Pickdisease (PD), fronto-temporal dementia (FTD), corticobasal degeneration(CBD) and progressive supranuclear palsy (PSP), in a subject.
 31. An invitro or ex vivo method for diagnosing or monitoring a disorder mediatedby neurofibrillary tangles, neuropil threads or dystrophic neurites,such as tauopathies, including Alzheimer's disease (AD), Pick disease(PD), fronto-temporal dementia (FTD), corticobasal degeneration (CBD)and progressive supranuclear palsy (PSP), in a subject, comprising thesteps of: a) contacting in vitro an appropriate biological sample fromsaid subject with a diagnostic agent of any one of claims 17-20 and23-26, and b) determining the presence or the absence ofphosphorylated-tau protein, in said biological sample, the presence ofsaid of phosphorylated-tau protein indicating that said subject has adisorder mediated neurofibrillary tangles, neuropil threads ordystrophic neurites, such as tauopathies, including Alzheimer's disease(AD), Pick disease (PD), fronto-temporal dementia (FTD), corticobasaldegeneration (CBD) and progressive supranuclear palsy (PSP).
 32. A invitro or ex vivo method for monitoring the progression or regression ofa disorder mediated by neurofibrillary tangles, neuropil threads ordystrophic neurites, such as tauopathies, including Alzheimer's disease(AD), Pick disease (PD), fronto-temporal dementia (FTD), corticobasaldegeneration (CBD) and progressive supranuclear palsy (PSP), in asubject, comprising the steps of: a) contacting in vitro an appropriatebiological sample from said subject with a diagnostic agent of any oneof claims 17-20 and 23-26, b) determining the amount ofphosphorylated-tau protein in said biological sample, and c) comparingthe amount determined in step (b) with the amount of phosphorylated-tauprotein previously obtained for said subject, a significant increase inamount of phosphorylated-tau protein constituting a marker of theprogression of said disorder mediated by neurofibrillary tangles,neuropil threads or dystrophic neuritis, and a significant decrease ofphosphorylated-tau protein constituting a marker of the regression ofsaid disorder mediated by neurofibrillary tangles, neuropil threads ordystrophic neurites.
 33. A method for in vivo imaging neurofibrillarytangles, neuropil threads or dystrophic neurites in a subject comprisingthe steps of a) administrating a detectable quantity of a diagnosticagent of any one of claims 17-20 and 23-26 in a subject, preferably ahuman and, b) detecting the diagnostic agent in said subject by animaging method.
 34. A pharmaceutical composition comprising atherapeutic agent of any one of claims 17-22 and 26, and apharmaceutically acceptable carrier.
 35. A method for obtaining a VHHdirected against a phosphorylated tau protein comprising a step ofimmunizing a camelid with the single phospho-peptide derived from theC-terminus of a tau protein of sequence CSIDMVDS(PO₃H₂)PQLATLAD (SEQ IDNO. 6) or a phospho-tau enriched Alzheimer's disease brain extract froma human.